The effect of removing anti-nutritional factors from n-hexane-extracted mustard meal using 80% isopropanol (to reduce thioglucosides, phenolics, etc.) on growth, food efficiency ratio, serum and liver lipid profiles and protein content of young rats was examined. For this n-hexane-extracted mustard meal was extracted with 80% isopropanol giving a fraction with 68% protein and low residual thioglucoside (0.5%) as well as phenolic (0.3%) content. This isopropanol-extracted mustard seed protein fraction reduced the growth of young rats slightly when compared with casein. The food efficiency ratio between rats fed isopropanol-extracted mustard seed protein or casein did not differ, nor did the protein composition affect serum total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol and LDL-C/HDL-C ratio. However, rats fed isopropanol-extracted mustard seed protein showed a significantly lower (p<0.05) liver cholesterol concentration than rats fed casein. Liver triglyceride and phospholipid concentrations did not differ between rats fed the two proteins, nor was serum protein affected. This study indicates that extraction of hexane-extracted mustard meal with 80% isopropanol reduced a number of anti-nutritional factors like thioglucoside and phenolics. Furthermore the nutritional quality of mustard seed protein fraction is comparable to casein in respect to growth, food efficiency ratio, serum lipid and protein concentrations and organ weights.