Selective serotonin reuptake inhibitors are used as first line treatment in major depressive disorder. However, selective serotonin reuptake inhibitors have also been associated with sexual disorders, abnormalities, and sexual dysfunction, although mechanisms are unclear. The aim of this project was to investigate the possible endocrine disrupting effect of sertraline (SER) on sex steroid production in male rats exposed to 3 therapeutically realistic doses of SER 1.25, 5, and 20 mg/kg/day. To achieve this, we analyzed all the major steroids in testis, adrenals, brain, and plasma using Liquid chromatography tandem mass spectrometry. Furthermore, we investigated the potential effects on gene expression on the major genes involved in testicular, adrenal and brain steroidogenesis using quantitative PCR. Moreover, plasma luteinizing hormone (LH) levels were analyzed. We observed significant reduction in steroid production, in particular on the testicular Δ-4 axis and on the adrenal CYP17-hydroxylase axis. Effects in brain and plasma were less pronounced. Testicular gene transcription was also significantly down-regulated except for the CYP19 (aromatase) gene. In contrast, no effects on the adrenal gene expression were observed, except for an up-regulation of the CYP17. Plasma LH and LH/TS were increased, in particular in the lowest exposure group, indicating some degree of compensatory hypogonadism. In conclusion, this study demonstrates extensive endocrine disruption during SER exposure in male rats, both directly on steroid production in major endocrine tissues, but also indirectly by affecting gene expression. Furthermore, increased LH levels may augment decreased sex steroid production, in particular testosterone production, inducing a state of compensatory hypogonadism.