Portulaca oleracea is a fleshy annual herb which is distributed throughout the temperate and tropical areas of the world. Its crude extracts have been reported to have deleterious effects on reproductive parameters in male rats. The crude extracts of this plant have been reported to have deleterious effects on reproductive parameters 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 on silica gel for fractionation. Out of the 5 fractions obtained, fraction 2 was then subjected to male rats’ reproductive bioassay. Twenty male rats were divided into control (distilled water) and fraction 2 (1, 2, 3 mg/kg) treated groups (5 per group) for hormonal assay and andrological studies. The animals were orally treated on daily basis for 50 days. Plasma testosterone level was assayed using Enzyme-Linked Immunosorbent Assay (ELISA) and semen analysis was done microscopically. Treatment of rats for 50 days with fraction 2 (1 mg/kg, 3mg/kg) caused significant (p<0.05) reductions in testosterone levels relative to the control. Fraction 2 (1 mg/kg, 2 mg/kg, 3 mg/kg) caused significant (p<0.05) decrease in sperm motility, sperm viability and sperm counts of rats relative to their respective controls. It can therefore be concluded that fraction 2 of Portulaca oleracea probably has deleterious effects on reproductive parameters in male rats.