Portulaca oleracea is a fleshy annual herb which is distributed throughout temperate and tropical areas of world. Crude extracts of this plant caused significant decreases in testosterone levels in male rats. Air-dried specimen of Portulaca oleracea was cold-extracted in methanol for 72 hours. The resulting methanol extract was then subjected to open column chromatography for fractionation. Out of 5 fractions obtained, fraction 1 was then subjected to male rats’ hormonal bioassays. Twenty male rats were divided into control and fraction 1 (1, 2, 3 mg/kg) treated groups for hormonal assay study. The animals were orally treated on daily basis for 50 days and allowed a recovery (withdrawal) period of 50 days after which hormonal assays were carried out at the end of dosing and recovery periods. Plasma LH, FSH and testosterone levels were assayed using Enzyme – Linked Immunosorbent Assay (ELISA) technique. Treatment of rats for 50 days with fraction 1 (1 mg/kg, 2 mg/kg, 3 mg/kg) caused significant (p<0.05) decreases in LH levels but caused no significant (p>0.05) changes in FSH levels relative to their respective controls. Fraction 1 (1 mg/kg, 2 mg/kg, 3 mg/kg) caused significant (p<0.05) increases in testosterone levels relative to control. There were significant (p<0.05) reductions in FSH levels of fraction 1 (1 mg/kg, 2 mg/kg and 3 mg/kg) recovery groups relative to control. It can therefore be concluded that fraction 1 of Portulaca oleracea probably induced significant changes on plasma gonadotrophins and testosterone levels through hypothalamic – pituitary testicular axis which could result in irreversible sterility.