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The effects of a Tween 80 and CaCl2 mixture on the hypocholesterolemic activity of bile salt hydrolase (BSH)-active Lactobacillus casei F0422 were investigated in rats fed a cholesterol-rich diet for 3 weeks. This mixture significantly enhanced the hypocholesterolemic activity of strain F0422 in rats. The group administered drinking water supplemented with both strain F0422 and the Tween 80 and CaCl2 mixture had significantly lower levels of total serum cholesterol, low-density lipoprotein cholesterol and total liver cholesterol than did the group administered drinking water supplemented only with strain F0422 or the Tween 80 and CaCl2 mixture. The hypocholesterolemic activity of the combined strain F0422 and Tween 80 and CaCl2 mixture was attributed to the increase in fecal bile acid excretion through enhancement of bile salt tolerance and BSH catalytic activity of strain F0422 by Tween 80 and up-regulation of the adhesion of strain F0422 to intestinal epithelial cells by CaCl2.