Purpose Acupuncture is an effective therapy for Internet addiction (IA). However, the underlying mechanisms of acupuncture in relieving compulsive Internet use remain unknown. Neuroimaging studies have demonstrated the role of the ventral striatum (VS) in the progress of IA; hence, the aim of this study was to explore the effects of acupuncture on the resting-state functional connectivity (rsFC) and relevant network of VS in IA. Methods Twenty-seven IA individuals and 30 demographically matched healthy control subjects (HCs) were recruited in this study. We acquired the functional magnetic resonance imaging (fMRI) data in IA subjects before and after 40 days of acupuncture treatment. Seed-to-voxel and ROI-to-ROI analyses were applied to detect the rsFC alterations of the VS and related network in IA subjects and to investigate the modulation effect of acupuncture on the rsFC. Results Compared with HCs, IA subjects exhibited enhanced rsFC of the right ventral rostral putamen (VRP) with the left orbitofrontal cortex (OFC), premotor cortex (PMC), cerebellum, and right ventromedial prefrontal cortex (vmPFC). In the network including these five ROIs, IA also showed increased ROI-to-ROI rsFC. Using a paired t-test in IA subjects before and after 40 days of acupuncture, the increased ROI-to-ROI rsFC was decreased (normalized to HC) with acupuncture, including the rsFC of the right VRP with the left OFC, PMC, and cerebellum, and the rsFC of the left cerebellum with the left OFC, PMC, and right vmPFC. Furthermore, the change in rsFC strength between the right VRP and left cerebellum in IA individuals was found positively correlated with the Internet craving alleviation after acupuncture. Conclusions These findings verified the modulation effect of acupuncture on functional connectivity of reward and habit systems related to the VS in IA individuals, which might partly represent the underlying mechanisms of acupuncture on IA.