Flurbiprofen is one of the nonsteroidal anti-inflammatory drugs. Whether flurbiprofen affects androgen biosynthesis in Leydig cells is still unknown. Immature Leydig cells (ILCs) isolated from 35-day-old male Sprague-Dawley rats were cultured with 0-100 μM flurbiprofen for 24 h and medium androgen levels and Leydig cell mRNA levels were measured. Immature Leydig cells were also incubated with 100 μM flurbiprofen for 3 h in combination with luteinizing hormone (LH), 8bromo-cAMP, 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively, and medium androgen levels were measured. The ROS generation and apoptosis rate were also investigated. The direct effects of flurbiprofen on androgen biosynthetic and metabolizing enzyme activities were measured. Flurbiprofen significantly inhibited basal, LH, and 8bromo-cAMP stimulated androgen production at 10 and 100 μM. Further study demonstrated that flurbiprofen competitively inhibited rat and human testis 3β-hydroxysteroid dehydrogenase (HSD3B) activity with the half maximal inhibitory concentration (IC50) values of 0.95 μM for rat enzyme and 6.31 μM for human enzyme. In addition, flurbiprofen down-regulated the expression of Srd5a1 and Akr1c14 at 1, 10, and 100 μM. Flurbiprofen also down-regulated Lhcgr expression at 100 μM. Flurbiprofen at 10 and 100 μM increased ROS production and apoptosis rate of rat Leydig cells. In conclusion, flurbiprofen directly inhibits HSD3B activity and the expression levels of Srd5a1 and Akr1c14 in rat Leydig cells, thus leading to the reduction of androgen secretion.