Abstract: Stephania glabra (Roxb.) Miers (Menispermaceae) is a large, climbing shrub, indigenous to lower Himalaya of India. This plant has long been used for the treatment of asthma, tuberculosis, dysentery, hyperglycemia, cancer, fever, intestinal complaints, sleep disturbances and inflammation in India. The study aim is to isolate the chemical constituents from the plant tubers and to evaluate antimicrobial activity of isolated constituents. Ethanolic extract from tubers was subjected to column chromatography using silica gel as adsorbent for isolation of chemical constituents. The agar diffusion method was adopted for antimicrobial activity to determine IZD and MICs. A tetrahydroberberine alkaloid named 8-(4’-methoxybenzyl)-xylopinine has been isolated from the column eluent of CHCl3:MeOH (1:9). The compound showed antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Eschericha coli and Salmonela typhi with IZD range of 8-19 mm at MIC of 25-100 μg mL-1. Erythromycin (10 μg mL-1) was used as standard antibiotic. Present study concludes that 8-(4’-methoxybenzyl)-xylopinine have significant antibacterial activity.