The applications of photo-fermentative bacteria (PFB) for continuous hydrogen production are generally subjected to a serious biomass washout from photobioreactor, resulting from poor flocculation of PFB. In this study, through reducing the absolute zeta-potentials of PFB, Ca2+ greatly decreased total interaction energy barrier of PFB based on DLVO theory, thus promoted the bioflocculation of Rhodopseudomonas faecalis RLD-53. Average floc size of PFB increased with the Ca2+ concentration, and reached maximum 30.07 mu m at 4 mmol/l. Consequently, biomass retention capacity of photobioreactor significantly enhanced after 30 min settling with half working volume discharge. In the continuous photo-fermentative sequencing batch reactor, compared with the free cell culture, bioflocculation reached a higher steady-state hydrogen production rate of 879 ml H-2/l/d and hydrogen yield of 2.64 mol H-2/mol acetate, respectively. Therefore, bioflocculation promoted by calcium ion was an effective strategy for retaining PFB in photobioreactor to produce hydrogen continuously. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.