This study investigated the effects of various arsenite concentrations on bio-hydrogen production from molasses using a sequence batch reactor (SBR) operated in a series of three batch cycles. In the first batch cycle, hydrogen production was stimulated at arsenite concentrations lower than 2.0 mg/L, while inhibition occurred at arsenite concentration higher than 2.0 mg/L compared to the control. Hydrogen production decreased substantially during the second batch cycle, while no hydrogen was produced during the third batch cycle at all tested concentrations. The toxic density increased with respect to the increase in arsenite concentrations (6.0 > 1.6 > 1.0 > 0.5 mg/L) and operation cycles (third cycle > second cycle > first cycle). The presence of microorganisms such as Clostridium sp. MSTE9, Uncultured Dysgonomonas sp. clone MEC-4, Pseudomonas parafulva FS04, and Uncultured bacterium clone 584CL3e9 resulted in active stimulation of hydrogen production, however, it was unlikely that Enterobacter sp. sed221 was not related to hydrogen production. The tolerance of arsenite in hydrogen producing microorganisms decreased with the increase in induction time, which resulted in severing the inhibition of continuous hydrogen production.