Compounds of natural origin are often used for their beneficial effects on the male endocrine system and the synthesis of steroid biomolecules in testicular tissue. One of such compounds is quercetin (QUE), which belongs to the flavonoid family and is found in a wide range of vegetables, fruits and plant products. The purpose of this study is to evaluate the impact of QUE on the endocrine function of rat testicular fragments under in vitro conditions. Testicular fragments from adult Wistar rats (n=9), cultured in the D-MEM medium with different concentrations of QUE (namely 1, 10 and 100 µmol/L) for 24 h at 37°C (5% CO2), were used in the experiment conducted. Following culture, the medium was separated and the levels of cholesterol (CHOL) and male hormones were measured. CHOL values were quantified spectrophotometrically, whereas the concentrations of androstenedione (ANDRO), dehydropeiandrosterone (DHEA) and testosterone (TEST) were quantified using the enzyme-linked immunosorbent assay (ELISA) commercial kit. The results obtained indicate that 10 µmol/L QUE significantly increased (P<0.001; P<0.05) the concentrations of all the steroid biomolecules considered (CHOL, ANDRO, DHEA and TEST) when compared to the control samples. Accordingly, our findings confirm the positive impact of QUE on the endocrine function and steroidogenesis of rat testicular tissue under in vitro conditions.