The effects of QB-binding D1-protein mutations on the phenotypic characteristics and on hydrogen production of sulfur-deprived Chlamydomonas reinhardtii P. A. Dang. cultures were investigated. The mutation involved one (D240) or double (D239–40) amino-acid deletions at positions 240 and 239–240, respectively, in the loop connecting helices D and E of the D1 protein. Phenotypic characterization of the mutants showed the following peculiarities as compared to the wildtype (WT): (i) a higher sensitivity to photoinhibition, (ii) a reduced amount of chl per dry weight and per cell, (iii) a higher respiration-to-photosynthesis ratio, (iv) a higher carbohydrate accumulation during the aerobic phase, and (v) a higher synthesis of xanthophyll-cycle pigments. These differences were translated into a 12- to 18-fold higher hydrogen biogas production.