An efficient protocol had been developed for the root culture of Luffa acutangula (L.) Roxb. The root and leaf explants from 21 day old in vitro raised seedlings were cultured on half strength Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of Indole-3-acetic acid(IAA), Indole-3-butyric acid (IBA) and Napthalene acetic acid (NAA). IBA at 1.0 mgl -1 combined with NAA (1.0 mgl -1 ) induced maximum percentage of rooting from leaf explants, under total dark condition. After 3 weeks, well established roots were separated. Fresh root tissue from the leaf explants and root tips from in vitro seedlings were subcultured in half strength MS liquid medium supplemented with different concentrations and combinations of IAA, IBA and NAA, under continuous agitation at 80 rpm under total dark condition. The biomass of root culture (derived from leaf explants) in the MS medium fortified with IBA(1.0 mgl -1 ), NAA(1.0mgl -1 ) and L-glutamine (20 mgl -1 ) was increased to 5.38 g FW and the biomass of root culture (derived from root tip) in the MS medium supplemented with IBA (0.5 mgl -1 ), NAA (1.0 mgl -1 ) and L-glutamine (20 mgl -1 ) was increased to 5.82 g FW after 7 weeks of culture.