Hypercholesterolemia has been reported to be the main cause of cardiovascular diseases and the leading cause of death. Therefore, decreasing serum cholesterol level is very important for preventing the cardiovascular diseases. It has been supposed that probiotics in human gastrointestinal tract have the ability to decrease serum cholesterol level by reducing the absorption of cholesterol from the intestinal tract and the bile salt deconjugation. In this study, 28 strains of Lactobacillus spp., isolated from breast-fed infant's feces, were identified and investigated for their bile salt deconjugation ability. The deconjugation ability of the strains was determined by the release of cholic acid resulting from the deconjugation of conjugated bile salts. Research results showed that four of the strains had bile salt deconjugation ability. The strains with deconjugation ability have been identified in species level by using biochemical test, and molecular techniques, API 50CHL test and 16S rRNA gene sequence analysis respectively. LP1, E3, and E9 strains with deconjugation activity were identified as Lactobacillus rhamnosus and GD2 strain as Lactobacillus plantarum. Even if oxgall decreases the viability of bacteria, the highest amount of cholesterol precipitation (42%) was performed by GD2 strain in the presence of 0.3% (w/v) bile. This study demonstrated that the identified Lactobacillus strains had an excellent ability to survive at low pH, a high bile deconjugation ability, and hypocholesterolemic effect in in vitro conditions.