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Background Formaldehyde, a ubiquitous environmental pollutant, is used extensively and has been proved to impair male reproduction in mammals. However, no trials have explored whether formaldehyde affects sexual function. Aim To evaluate the effect of long-term formaldehyde exposure on sexual behavior and to investigate the potential mechanism. Methods Forty C57BL/6 male mice were randomly allocated to four equally sized groups. Mice were exposed to formaldehyde at a dose of 0 (control), 0.5, 5.0, or 10.0 mg/m 3 by inhalation for 60 days. Outcomes Sexual behavior, body and reproductive organ weights, testosterone concentration in serum and testicular tissue, expression of steroidogenic enzymes, quality of sperm, and testicular structure were measured. Results Formaldehyde inhibited sexual behavior and decreased reproductive organ weights in mice. Serum testosterone levels and intratesticular testosterone concentrations were decreased in the formaldehyde-treated groups. Expression levels of steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase (3β-HSD), also were decreased in the testes of mice exposed to formaldehyde. Moreover, the structure of seminiferous tubules was destroyed and sperm quality decreased after formaldehyde exposure. In addition, the results indicated that the effects of formaldehyde were dose dependent. Clinical Implications Efforts should be undertaken to decrease impairment of sexual function caused by formaldehyde exposure. Strengths and Limitations The relatively small sample might have affected the outcomes. Further experiments are needed to study the mechanism of action of formaldehyde. Conclusion Exposure to formaldehyde gas inhibited sexual behavior, caused reproductive organ atrophy, and impaired spermatogenesis in male mice, which might have been induced by suppressed expression of steroidogenic enzymes in Leydig cells and decreased testosterone synthesis. Zang Z-J, Fang Y-Q, Ji S-Y, et al. Formaldehyde Inhibits Sexual Behavior and Expression of Steroidogenic Enzymes in the Testes of Mice. J Sex Med 2017;14:1297–1306 . [ABSTRACT FROM AUTHOR]