Eurycoma longifolia Jack, a small Simaroubaceae tree, known locally as 'Tongkat Ali' is popularly used as a sexual tonic in traditional medicine for aphrodisiac activity and improvement of fertility and male libido. To investigate the effects of the standardized bioactive fraction of E. longifolia and its chemical constituents on the male fertility and the mechanisms of action involved. The powdered roots of E. longifolia were extracted separately with methanol and water. The organic extract upon further fractionation on HP 20 resin and elution with the methanol/water mixture afforded four fractions (F1-F4). These fractions, together with the crude aqueous (W) and organic extracts were standardized following their respective major quassinoid content and profile. The effects of the fractions on the rat spermatogenesis were compared with that of the aqueous extract (W) to determine the bioactive fraction. The effects of the bioactive fraction on the sperm count and quality, the histological morphometric changes on the spermatogenesis cycle, fertility and hormonal changes of plasma testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and estrogen in the animals upon oral administration were determined. The effects of the bioactive quassinoids on the testosterone release from the isolated testicular interstitial cells rich in Leydig cells, were also described. The male rats orally administered with 25mg/kg of F2 and 250mg/kg of W, significantly increased the sperm concentration when compared with that of the control animals (P<0.05). High performance liquid chromatography analysis revealed that 25mg/kg of F2 and 250mg/kg of W were almost similar in concentration of eurycomanone, the major and most potent quassinoid. Microscopic morphometrical analysis of the rat testis following treatment with F2, showed significant increase in the number of spermatocytes and round spermatids at Stage VII of the spermatogenesis cycle when compared to that of the control (P<0.05). The estimated spermatozoa production rate and the number of Leydig cells were also elevated (P<0.001). The fertility index, fecundity index and the pup litter size delivered from the females after mating with the males treated with F2 were increased. The plasma testosterone level of the animals given 25mg/kg of F2 orally was significantly different at day-26 (p<0.05) and day-52 (P<0.01) from those of control but was not different at day-104. The testicular testosterone also peaked in the animals treated with 25mg/kg F2 and was higher than that in the plasma. The plasma LH and FSH levels of the rats treated with 25mg/kg of F2 were higher than those of the control (P<0.001). In contrast, the plasma estrogen level was significantly lower than that of the untreated control. Amongst the isolated quassinoids of F2, eurycomanone and 13α(21)-dihydroeurycomaone significantly increased the testosterone level from the Leydig cells of the testicular interstitial cells cultured in vitro (P<0.05). The standardised extract F2 of E. longifolia and its major quassinoids especially eurycomanone improved the rat spermatogenesis by affecting the hypothalamic-pituitary-gonadal axis and the potential efficacy may be worthy of further investigation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.