Abstract: Clostridium butyricum (C. butyricum) is a probiotic. C. butyricum of human origin was obtained by a combination of selective media screening, morphological identification, and molecular biological identification. Its probiotic properties such as antioxidant capacity and lipid-lowering capacity were investigated experimentally. One strain named C. butyricum B-3 was screened from the faeces of 33 healthy volunteers. A commercial strain, C. butyricum MIY588, was introduced as a positive control. Moreover, their probiotic properties were evaluated, including simulated digestive tract tolerance, bile salt tolerance, antibiotic sensitivity, antioxidant capacity, and lipid-lowering capacity by in vitro simulation tests. The results showed that C. butyricum B-3 was highly tolerant to the simulated digestive tract environment and bile salts, with a survival rate of 94.47% in simulated artificial gastric fluid, 82.11% in simulated artificial intestinal fluid, and higher than 89% in high concentrations of bile salts. It was sensitive to penicillin, ampicillin, norfloxacin, cyclofloxacin, gentamicin, amikacin, and resistant to cotrimoxazole and erythromycin and cefazolin. The intact strains (IS) of C. butyricum B-3 had the highest hydroxyl radical scavenging rate at around 80%. The fermentation supernatants (FS) of both strains had comparable DPPH radical scavenging and reducing power capacities, which were significantly higher than those of IS and cell-free extract (CFE) (P<0.05). The binding capacity of IS for both bile salts was higher than that of FS for both strains. The inhibition of pancreatic lipase by FS for C. butyricum B-3 was lower than that of the control strain, but the inhibition of pancreatic lipase by IS was significantly higher than that of the control strain (P<0.05) at 44.02%. The cholesterol scavenging capacity of C. butyricum B-3 was 21.07%, which was slightly lower than that of the positive control strain. The above results indicate that C. butyricum B-3 has the potential to be an excellent probiotic and has great exploitation value in the research and development of functional foods.