Production of freshwater catfish (Mystus numerus Cuv. & Val.) has soared to meet consumer demand, but little is known about the nutritional requirements of the species. This study was conducted to determine the dietary lysine requirement of juvenile Mystus numerus fed semipurified diets containing graded levels of lysine. The control diet contained fish meal, gelatin and crystalline amino acids simulating the amino acid profile of Mystus nemurus muscle protein except for lysine. Seven experimental diets were formulated to contain lysine as 0.7% (2.00%), 1.1% (3.14%), 1.5% (4.29%), 1.9% (5.43%), 2.3% (6.57%), 2.7% (7.71%) and 3.1% (8.86%) of the diet (% of protein). Triplicate groups of fish were fed to satiation for 90 days. Specific growth rate, feed efficiency ratio, protein efficiency ratio, productive protein value and daily net protein retention of fish fed diets containing lysine at 2.00% of protein were significantly lower than fish fed diets containing lysine at higher levels. Plasma ammonia concentrations were similar in all groups of fish. A digestibility trial between fish fed the experimental diet with 7.71% of lysine and a diet with fish meal as the sole source of protein showed that protein digestibility of the experimental diet was significantly higher than that of the fish meal based diet. Non-linear regression analysis of specific growth rate against dietary lysine levels indicated a lysine requirement of 3.47% of protein for this species.