Objective <p>The relationship between sedentary behavior and infertility remains ambiguous and contentious. This study seeks to elucidate this association by analyzing data from the 2013–2018 National Health and Nutrition Examination Survey (NHANES), coupled with Mendelian randomization (MR) analyses.</p> Methods <p>Our analysis comprised 2904 female participants, aged 20 to 49 years, enrolled in the National Health and Nutrition Examination Survey (NHANES) during the 2013–2018 cycles. Weighted multivariate logistic regression model was employed to examine the association between sedentary behavior and infertility, with sensitivity analysis conducted to validate the robustness of the findings. In addition, we used restricted cubic spline (RCS) curves to explore any non-linear association between sedentary behavior and infertility. A two-sample Mendelian randomization (MR) analysis was subsequently conducted using summary-level data from genome-wide association studies (GWAS) to investigate the potential causal links between self-reported leisure screen time (LST), sedentary commuting, sedentary behavior at work, and infertility. Causal estimates were primarily obtained with the inverse-variance weighted (IVW), while the weighted median, MR-Egger, and weighted mode were applied as complementary analyses. To evaluate the robustness of these results, horizontal pleiotropy was assessed using the MR-Egger intercept, heterogeneity was examined with Cochran’s Q test, and additional sensitivity testing was performed through leave-one-out analyses.</p> Results <p>After adjusting for potential confounders, the weighted multivariable logistic regression analysis indicated that although the prevalence of infertility appeared to increase with longer daily sitting time, this association did not reach statistical significance (OR = 1.03, 95% CI: 1.00–1.07, <i>P</i> = 0.066). Results from multiple sensitivity analyses remained largely consistent, supporting the robustness of these findings. In the Mendelian randomization (MR) analysis, no statistically significant causal relationship was observed between genetically predicted sedentary behavior and infertility. Specifically, the inverse variance–weighted (IVW) estimates suggested no robust evidence of causality between leisure screen time (OR = 1.11, 95% CI: 0.10–1.24, P = 0.052), sedentary commuting (OR = 1.19, 95% CI: 0.88–1.62, P = 0.257), or sedentary behavior at work (OR = 0.99, 95% CI: 0.83–1.19, P = 0.930) and infertility.</p> Conclusion <p>No statistically significant evidence was found to support an association between sedentary behavior and infertility. Future large-scale prospective studies are warranted to further explore this potential relationship.</p>