The aim of this study was to understand the enhancement mechanism of H-2 photoproduction in Chlorella protothecoides under simultaneous nitrogen limitation and sulfur deprivation (LNS). Nitrogen limitation (LN) rather than sulfur deprivation significantly inhibited relative variable fluorescence at K-step (WK) and J-step (Vi), photochemical efficiency of PSII (photosystem II), F-v/F-m, during the process of incubation in the light. Under such conditions, photosynthetic O-2 evolution decreased and the anaerobiosis was established after 12 h of incubation. The algae generated large amounts of H-2 under nitrogen limitation but generated only trace amounts under sulfur deprivation. Obviously, nitrogen limitation rather than sulfur deprivation was the decisive factor that induced H-2 photoproduction in C. protothecoides under LNS. The LNS culture generated much more H-2 than the LN culture in the presence of DCMU during incubation, suggesting that a PSII-independent electron source contributed many more electrons for transfer to hydrogenase in the LNS culture. PSII electron transport includes linear electron flow (LEF) and cyclic electron flow (CEF) of PSII in C. protothecoides. In the PSII-dependent electron source for H-2 photoproduction, PSII supplies electrons to hydrogenase through the LEF. The LNS culture showed much higher LEF and lower CEF than the LN culture during the H-2 photoproduction phase, as indicated by the large lower quantum yield of PSII electron transport (Phi(PSII)) in the LNS culture in the presence of DCMU. Therefore, compared with nitrogen limitation, simultaneous nitrogen limitation and sulfur deprivation enhanced H-2 photoproduction in C. protothecoides mainly due to enhanced PSII-dependent and -independent electron sources. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.