Biohydrogen is a clean and efficient source of energy produced easily by anaerobic systems. Therefore, the discovery of novel and efficient production methods and utilization of inexpensive starting material are crucial for economical biohydrogen production. In this study, novel hydrogen producing bacterial strain Clostridium sp. SH25 was screened from the anaerobic sludge obtained from a water treatment plant, which showed a higher hydrogen-producing activity on glycerol than other strains. The effective hydrogen production was evaluated under varying anaerobic culture conditions, and the optimum temperature, initial pH, additional NaCl concentration, and inoculum size were 37 ℃, 6.0, 0%, and 10% (v/v), respectively. The cumulative hydrogen production volume from crude glycerol was 24.30±1.07ml after 36 h. To test the practical application of biohydrogen, a 20ml culture of Clostridium sp. SH25 was incubated for 12 h and directly applied to a small hydrogen car unit operated for 19.05±0.33 s with 8.37±0.21m displacement. Overall, identification of the efficient Clostridium sp. SH25 strain resulted in the production of a large amount of biohydrogen, which further supported the operation of a small hydrogen car. This implied a possible application of biosystems in biohydrogen production.