This study investigated the effects of initial pHs and temperatures to the hydrogen production ration and cell growth of Biohydrogenbacterium R3 sp.nov.. The initial pHs were set at 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 6.7, respectively and the temperatures were increased from 25 °C to 45 °C in regular intervals of 5 °C at 10 g/L of the initial glucose concentration. The results indicated that pH value had a noticeable effect on the cell growth and hydrogen production. The dry cell weight and hydrogen production yield got the maximum of 0.6308 g/L and 34.2 mmol/L, respectively when the initial pH was 5.5. The final pH in the culture were always kept at 3.0~4.0. Temperature is also known to affect the maximum cell growth and specific hydrogen production ration (SHPR). And they got the maximum of 0.6682 g/L and 1.0145mol H2/mol glucose, respectively when the temperature was 30 °C. It is obvious that hydrogen production and biomass will be inhibited gradually with increasing this pH and temperature or decreasing it.