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Abstract Background Cholesterol removal by isolated Lactococcus lactis subsp. lactis was investigated in vitro. The studied mechanisms were (1) assimilation of cholesterol by growing bacterial cells, (2) deconjugation of bile salts by bile salt hydrolase, (3) precipitation of cholesterol with deconjugated bile, (4) binding of cholesterol to the cell membrane, and (5) incorporation of cholesterol into the cell membrane. Result Levels of cholesterol assimilated by growing cells (43.70%) were significantly higher than those assimilated by resting cells (12.93%) or counterpart dead cells (6.35%). Scanning electron micrographs indicated that cholesterol adsorbed to the bacterial cell wall. A comparison of IR spectra of Lactococcus lactis subsp. lactis grown with and without cholesterol using an FTIR spectroscopy database revealed intensity changes associated with sulfamide bonds, P–H stretching and the C≡C bonds. Precipitation of cholesterol upon deconjugation of glycocholate was higher (5.53 μg/ml) than that observed upon taurocholate (3.7 μg/ml). Conclusions The present study suggested the ability of probiotic Lactococcus lactis subsp. lactis to reduce its cholesterol level through several different mechanisms. Consequently, this probiotic bacterium showed an interested potential of application in cholesterol reduction to be a novel and save prospect in resolving hypocholesteremia and related diseases.