Context: Illicit use of anabolic androgenic steroids (AAS) is frequently observed in men and is associated with subsequent testosterone deficiency although the long-term effect on gonadal function is still unclear. Serum insulin-like factor 3 (INSL3) has been suggested to be a superior biomarker of Leydig cell secretory capacity compared to testosterone. Objective: This study aimed to investigate serum INSL3 concentrations in AAS users. Methods: This community-based, cross-sectional study included men aged 18 to 50 years, involved in recreational strength training and allocated to 1 of 3 groups: never-AAS users as controls (n = 44), current (n = 46), or former AAS users (n = 42) with an average duration since AAS cessation of 32 (23; 45) months. Results: Serum INSL3 was lower in current AAS users and former AAS users than in controls, median (interquartile range), 0.04 μg/L (nondetectable [ND]-0.07 μg/L) and 0.39 μg/L (0.24-0.62 μg/L) vs 0.59 μg/L (0.45-0.72 μg/L), P less than. 001. Former AAS users exhibited lower serum INSL3 levels than controls in a multivariable linear regression even after adjusting for serum total testosterone (TT) and other relevant confounders, (B) (95% CI), -0.16 μg/L (95% CI, -0.29 to -0.04 μg/L), P equal to. 011. INSL3 and TT were not associated in the model, P equal to. 821. Longer accumulated AAS duration (log2) was associated with lower serum INSL3 in former AAS users, (B) (95% CI), -0.08 (95% CI, -0.14 to -0.01), P equal to. 022. Serum INSL3, but not inhibin B or testosterone, was associated with testicular size in a multivariate linear regression, (B) (95% CI); 4.7 (95% CI, 0.5 to 8.9), P equal to. 030. Conclusion: Serum INSL3 is reduced years following AAS cessation in men, independently of testosterone, suggesting persistently impaired Leydig cell capacity.