Sex disparities in inflammation have been reported, but the cellular and molecular basis for these discrepancies is unknown. Monocytes are central effector cells in immunity and possess high capacities to produce proinflammatory leukotrienes (LTs). Here, we investigated sex differences in the activation of 5‐lipoxygenase (5‐LO), the key enzyme in LT biosynthesis, in human peripheral monocytes. In cells from females, 5‐LO product formation was 1.8‐fold higher than in cells from males, as evaluated by HPLC. When female monocytes were resuspended in plasma from males, 5‐LO products were significantly lower than in female plasma. Interestingly, 5α‐dihydrotestosterone (5α‐DHT, 10 nM) repressed LT synthesis in female cells down to the levels observed in males, while estradiol (100 nM) was without effect, and progesterone (100 nM) caused only a slight inhibition. 5α‐DHT (10 nM) caused ERK phosphorylation and inhibition of phospholipase D (PLD), as evaluated by Western blot and measurement of PLD activity via radioenzymatic diacylglyceride (DAG) and nonradioactive choline assays. Accordingly, PLD activity and DAG formation were 1.4‐ to 1.8‐fold lower in male vs. female monocytes connected to increased ERK phosphorylation. Our data indicate that ERK activation by androgens in monocytes represses PLD activity, resulting in impaired 5‐LO product formation due to lack of activating DAGs.—Pergola, C., Rogge, A., Dodt, G., Northoff, H., Weinigel, C., Barz, D., Rådmark, O., Sautebin, L., Werz, O. Testosterone suppresses phospholipase D, causing sex differences in leukotriene biosynthesis in human monocytes. FASEB J. 25, 3377–3387 (2011). www.fasebj.org