Chlamydomonas reinhardtii is a green algae that can use light energy and water to produce hydrogen under anaerobic condition. This work reports the effect of limiting factors on hydrogen production in sulfur deprived anaerobic C. reinhardtii culture. In order to confirm the relationship between hydrogen production and limiting factors such as residual PSII activity and endogenic substrate degradation, the increase in chlorophyll concentration and the decrease in starch concentration was investigated during sulfur deprivation. The overall hydrogen production increased depending on cell density in range of $0.4{sim}0.96;g$ DCW/l. At this time, the increase in chlorophyll concentration during 24 h after sulfur deprivation increased in proportion to hydrogen production, however, the decrease in starch concentration was not proportional to that. Therefore, hydrogen production under sulfur deprivation using green alga was closely associated with the residual PSII activity than the endogenic substrate degradation.