The objective of this experiment was to study the effects of dietary cottonseed protein and casein on plasma and biliary lipids, plasma amino acids and gallstones in hamsters. Thirty‐four male hamsters (60 ± 5 g) were fed either the lithogenic “Dam Diet” (containing 20% casein, 74.3% sucrose and 5.7% vitamin‐mineral mix) or a similar diet that contained 20% cottonseed protein for 30 days. Both diets contained protein as a protein isolate. The concentration of alpha‐aminobutyric acid was significantly elevated in the casein‐fed group. Significant differences in the total plasma cholesterol or lipoprotein cholesterol concentrations were not observed between the two dietary groups. A significant elevation in the absolute concentration of biliary cholesterol was observed in the casein‐fed hamsters. Cottonseed protein‐fed animals exhibited a significantly elevated concentration of bile acids. The ratio of glycochenodeoxycholic:glycocholic acid was significantly higher in the cotton‐seed protein‐fed group. This study reports that an elevated concentration of biliary cholesterol with a concomitant decrease in bile acid concentration yields a condition favorable to gallstone formation. It is proposed that cottonseed protein may have a specific effect on the bile acid pool by increasing the ratio of glycochenodeoxycholic acid:glycocholic acid which, in turn, prevents formation of cholesterol gallstones.