A novel optical fiber-based photobioreactor was utilized to produce H 2 by indigenous purple nonsulfur bacterium Rhodopseudomonas palustris WP3–5 using acetate as the sole carbon source. Plastic cladding of conventional end-light optical fibers was removed to obtain side-light optical fibers (SLOF), which was inserted into photobioreactors as the internal light source. External irradiation by conventional lamps may also be provided for the bioreactor as supplemental light sources. The H 2 production performance and light conversion efficiency of the photobioreactor were assessed when various illumination systems were used. The light sources examined included SLOF excited by halogen lamp (OF-HL), SLOF excited by metal–halide lamp (OF-MH), tungsten filament lamp (TL), halogen lamp (HL), and binary combinations of the above. Compared with bioreactors illuminated by external lamps, the OF-HL system produced more H 2 (625 ml), had higher light conversion efficiency (1.80%), and achieved higher H 2 yield (1.19 mol H 2/mol acetic acid). However, among the single light sources examined, HL gave the highest overall ( ν H 2 ) and specific ( ν s, H 2 ) H 2 production rate of 8.68 ml/l h and 3.01 ml/h g cell, respectively, primarily due to enabling better cell growth. Using OF-MH system resulted in poor H 2 production, indicating that emission spectrum of light sources was critical to photo-H 2 production. Combination of two different light sources appeared to further enhance photo-H 2 production, especially when optical fibers and external lamps were combined. Combination of OF-HL and TL exhibited the highest H 2 yield, ν H 2 , and ν s, H 2 of 2.64 mol H 2/mol acetic acid, 17.06 ml/h l, and 9.47 ml/h g cell, respectively. However, the highest total H 2 production (944 ml) and light conversion efficiency (1.42%) were attained when two types of optical fibers were incorporated (i.e., the OF-HL/OF-MH system).