ObjectiveTo evaluate the effect of acupuncture on anisometropic amblyopia (AA) and explore its potential mechanism for children with AA. MethodsSeventy-six children with monocular AA were randomized into a conventional treatment group and a combined treatment group of acupuncture and conventional treatment, with 38 patients in each group. In the conventional treatment group, on the base of patching regimen, the red flashing, grating and visual stimulation were delivered. Each approach lasted for 5 min per session and was given once every two days, three times a week, for 4 consecutive weeks. In the acupuncture-combined treatment group, on the base of the regimen of conventional treatment group, acupuncture was applied to Jingming (BL1), Cuanzhu (BL2), Fengchi (GB20) and Guangming (GB37), with needles retained for 20 min per session, once every two days, three times of intervention a week and for 4 consecutive weeks. Before and after treatment, the best corrected vision acuity (BCVA) was observed to evaluate the clinical effect in the two groups. Before treatment started, 15 patients with AA on left side were randomized selected from each group and the resting-state functional magnetic resonance imaging (rs-fMRI) was operated before and after treatment. Simultaneously, eight healthy children with normal visual acuity were recruited to be the normal control group and received one-time rs-fMRI scanning. Based on the activation likelihood estimation (ALE), the visual “what” pathway network was constructed. By analyzing the amplitude of low frequency fluctuation (ALFF) and the regional homogeneity (ReHo), the differences in the regional autonomous function activities in the key brain areas of the “what” pathway were compared among the three groups. Results(1) Clinical effect: After treatment, BCVA was (0.6 [0.5, 0.6]), higher than that (0.4 [0.3, 0.5]) before treatment, presenting the statistical difference (P < 0.05) in the conventional treatment group. BCVA was (0.6 [0.6, 0.8]), higher than that (0.4 [0.4, 0.5]) before treatment, presenting the statistical difference (P < 0.05) in the acupuncture-combined treatment group. BCVA in the acupuncture-combined treatment group was higher than that of the conventional treatment group (P < 0.05) after treatment. The total effective rate was 86.1% in the acupuncture-combined treatment group, higher than that (65.8%) of the conventional treatment group (P < 0.05). (2) Mechanism: ① ALFF: In comparison with the healthy control group, the ALFF in the primary visual cortex of the “what” pathway for AA children was reduced significantly (P < 0.05), and ALFF in the right fusiform gyrus and the right inferior temporal gyrus increased significantly when compared with the healthy children (P < 0.05). Compared with that before treatment, there was no significant difference in the brain areas of the “what” pathway after treatment in the conventional treatment group (P > 0.05). In the acupuncture-combined treatment group, ALFF in the right inferior occipital gyrus and the right fusiform gyrus of the “what” pathway increased after treatment when compared with that before treatment (P < 0.05). After treatment, in comparison with the conventional treatment group, there was no significant difference in the brain areas of “what” pathway in the acupuncture-combined treatment group (P > 0.05). ② ReHo: ReHo in the right inferior occipital gyrus, the right fusiform gyrus and the right inferior temporal gyrus in the “what” pathway was elevated significantly in AA children when compared with that in the healthy control group (P < 0.05). ReHo of the left inferior occipital gyrus and the left fusiform gyrus increased significantly after treatment when compared with that before treatment in the conventional treatment group (P<0.05). ReHo of the right inferior occipital gyrus, the left and the right fusiform gyrus increased significantly after treatment when compared with that before treatment in the acupuncture-combined treatment group (P < 0.05). After treatment, ReHo of the right inferior temporal gyrus in the “what” pathway in the acupuncture-combined treatment group was significantly higher than that of the conventional treatment group (P < 0.05). ConclusionThe acupuncture combined with conventional treatment improves BCVA and the total effective rate of AA children. The changes in the regional function activity of the brain areas within the “what” pathway were dominated by the compensatory increase of the autonomous activity in the occipital lobe (inferior occipital gyrus) and the temporal lobe (inferior temporal gyrus) in AA children. Both conventional treatment and acupuncture can enhance the intensity of autonomous function activities in the occipital lobe (inferior occipital gyrus) and the temporal lobe (fusiform gyrus / inferior temporal gyrus) in the “what” pathway so as to relieve visual impairment. ReHo in the inferior temporal gyrus in the acupuncture-combined treatment group was higher than that of the conventional treatment group and it suggests that the inferior temporal gyrus may be the key brain area to the improvement of visual function in the “what” pathway.