eHealth literacy is critical for evaluating abilities in locating, accessing, and applying digital health information to enhance one's understanding, skills, and attitudes toward a healthy lifestyle. Prior research indicates that enhancing eHealth literacy can improve health behaviors such as physical activity (PA). Physical literacy (PL) refers to the ability to develop sustainable PA habits, taking into account various aspects of an individual. Notably, university students have shown a decline in PA and possess low PL levels. However, the connection between eHealth literacy and PL in this demographic has not been extensively studied, and it remains uncertain whether PA acts as a mediator between eHealth literacy and PL. This study examines the extent to which PA mediates the link between eHealth literacy and PL in Chinese university students and explores gender differences in these variables. In February 2022, a cross-sectional survey was administered to 1210 students across 3 universities in China. The instruments used were the Perceived PL Instrument, the International Physical Activity Questionnaire, and the Chinese version of the eHealth Literacy Scale. Correlations between eHealth literacy, PA, and PL were analyzed using Pearson product-moment correlation and multiple linear regression, while mediation models helped elucidate the interactions among the 3 variables. The response rate for the study was 92.9% (1124/1210). In the mediation analysis, eHealth literacy showed a significant direct effect on PL, with a coefficient of 0.78 (β .75, SE 0.02; P<.001). Moderate to vigorous physical activity (MVPA) accounted for 2.16% of the total effect, suggesting that MVPA partially mediates the relationship between eHealth literacy and PL. Additionally, male students outperformed female students in terms of MVPA (t636=4.94; P<.001) and PL (t636=3.18; P<.001), but no significant differences were found in eHealth literacy (t636=1.23; P=.22). The findings indicate that MVPA serves as a mediator in the link between eHealth literacy and PL among university students. Students with low eHealth literacy or limited PA are less likely to be physically literate. Thus, eHealth literacy plays a crucial role in enhancing PL and PA, especially when interventions targeting PL are implemented. Our results also suggest a need for targeted health education interventions aimed at improving MVPA and PL among female students, while also recognizing that eHealth literacy is comparable across genders at universities.