Background <p>The digital transformation in healthcare has led to an increase in the use of telemedicine and artificial intelligence (AI)based applications. This study aims to examine the associations between e-health literacy, attitudes toward AI, and acceptance of telemedicine, and to test whether attitudes toward AI statistically mediate the association between e-health literacy and acceptance of telemedicine.</p> Methods <p>Our study was conducted between January 1 and March 1, 2026. This cross-sectional study included 277 participants who visited a family health center. Data were collected using a sociodemographic information form, the General Attitude Scale toward Artificial Intelligence in Health Services, the Attitude Scale toward Telemedicine, and the e-Health Literacy Scale. Hierarchical regression analysis and mediation analysis using the bootstrap method (5,000 resamples) were performed.</p> Results <p>The mean age of the participants was 30.52 ± 9.95, and 57.4% were female. In the hierarchical regression analysis, e-health literacy (β = 0.28, <i>p</i> &lt; .001) and a positive attitude toward AI (β = 0.23, <i>p</i> = .010) were found to positively predict telemedicine acceptance, while a negative attitude toward AI (β = -0.28, <i>p</i> = .002) was found to negatively predict it. In the mediation analysis, it was determined that e-health literacy has a strong direct effect on telemedicine acceptance (β = 0.545, <i>p</i> &lt; .001), but AI attitudes do not play a mediating role in this relationship (<i>p</i> &gt; .05).</p> Conclusion <p>E- health literacy is a strong and direct determinant of telemedicine acceptance, independent of attitudes toward AI. Negative attitudes toward AI were associated with lower acceptance of telemedicine services. The results of our study suggest that strategies aimed at improving e- health literacy and addressing AI related concerns may be effective in promoting the widespread adoption of telemedicine, particularly in primary care settings.</p>