Abstract The efficiency of hydrogen production by different cyanobacterial species depends on several external factors. We report here the factors enhancing hydrogen production by filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005. Cells adapted to dark-anaerobic conditions produced hydrogen consistent with increased hydrogenase activity when supplemented with Fe2+. Stimulation of hydrogen production could be achieved by addition of reductants, either dithiothreitol or β-mercaptoethanol with higher production observed with the latter. Additionally, Fe2+ and β-mercaptoethanol added to nitrogen- and sulphur-deprived cells significantly stimulated H2 production with maximal value of 5.91 ± 0.14 μmol H2 mg Chla−1 h−1. Glucose and a small increase of osmolality imposed by either NaCl or sorbitol enhanced hydrogen production. High rates of hydrogen production were obtained in cells adapted in nitrogen-deprived medium with neutral and alkaline external pH, significant decrease of hydrogen production occurred under acidic external pH.