Cannabis use has been hypothesized to alter endocrine function. To investigate this hypothesis, we performed an extended steroid profiling in cannabis consumers and matched controls. Using LC-MS/MS, 70 endogenous steroids were reliably identified in serum. Seven major steroids were subject to absolute quantification.Multivariate analyses revealed a global increase in androgen levels among cannabis consumers. Androstenedione (A4), testosterone (T), and dihydrotestosterone (DHT) were among the most significantly increased steroids. In contrast, C11-oxy androgens showed no significant change. This pattern suggests that phytocannabinoids might selectively affect gonadal androgen synthesis without altering adrenal or peripheral pathways, possibly via direct effects on the testes, or disruption of the hypothalamic–pituitary–gonadal (HPG) axis function.Additionally, two progesterone metabolites, 11beta-hydroxyprogesterone (11B-OHP4) and 5beta-dihydroprogesterone (5B-DHP4), were markedly elevated in cannabis consumers. When the cannabis user group was stratified according to the corresponding usage biomarkers, it was shown that 11B-OHP4 could be a biomarker of general exposure, whereas 5B-DHP4 displayed a dose-dependent relationship.These findings highlight the value of extended steroid profiling for investigating hormonal variations and evidence a possible link between cannabis consumption and altered male endocrine function.