While there is strong evidence showing that many food-borne probiotics regulate cholesterol metabolism, few studies have examined how probiotics of human origin affect cholesterol metabolism. Because people living in so-called 'longevity villages' are unlikely to have hypercholesterolemia, we hypothesized that probiotics isolated from the residents would have cholesterol-reducing effects on rats with hypercholesterolemia. We isolated 16 strains of Lactobacillus from four longevity populations in China. The strains were tested in vitro for bile salt hydrolase (BSH) activity and two isolates, Lactobacillus reuteri A9 and Lactobacillus mucosae A13, were screened out. These two strains were then administered daily for 28 d to rats fed a cholesterol-rich diet. The serum total cholesterol levels in the L. reuteri A9 and L. mucosae A13 groups decreased by 24.3% and 21.6%, respectively. The serum low density lipoprotein cholesterol levels decreased by 23.8% and 25.2%, respectively. The L. reuteri A9 and L. mucosae A13 groups also exhibited upregulated hepatic mRNA expression of Sterol regulatory element-binding protein 2 (Srebp2) by 2.71-fold and 2.54-fold, respectively. The mRNA expression levels of hepatic low-density lipoprotein receptor (Ldlr) in the two groups were significantly up-regulated by 1.28-fold and 2.17-fold, respectively. The composition of gut microbiota was recovered by oral gavage in both experimental groups, and the destroyed diversity of gut microbiota was relieved.