Clinical investigations have indicated women have higher levels of adipocyte fatty acid binding protein (A-FABP) than men. The present study aimed to identify factors related to gender difference in serum A-FABP levels. A total of 507 participants (194 men, 132 premenopausal women, and 181 postmenopausal women) were enrolled in the present study. Serum A-FABP levels increased in the order from men to premenopausal women to postmenopausal women in both body mass index categories (<25.0 and ≥25.0 kg/m(2); all P < 0.05). Multiple stepwise regression analyses showed that after adjustment for factors related to serum A-FABP levels, the trunk fat mass was an independent and positive factor of serum A-FABP levels. For men, total testosterone was associated independently and inversely with serum A-FABP levels. For pre- and postmenopausal women, bioavailable testosterone and total testosterone were independent and positive factors associated with serum A-FABP levels, respectively. The present study demonstrated that the androgen was correlated with the serum A-FABP levels negatively in men, but positively in women. With these effects on the fat content, especially trunk fat, androgen might contribute to the gender difference in serum A-FABP levels.