Impacts of operation timing of feeding and withdrawal on anaerobic wastewater treatment utilizing purple non-sulfur bacteria have been investigated in mixed culture condition with acidogenic bacteria. Simulated wastewater containing glucose was treated in a laboratory-scale chemostat reactor, changing the timing of wastewater feeding and withdrawal. Rhodopseudomonas palustris, which does not utilize glucose as a substrate, was inoculated in the reactor. Rps. palustris was detected by a fluorescent in situ hybridization (FISH) technique using the specific Rpal686 probe. As a result, population ratios of Rps. palustris were over 20% through the operation. Rps. palustris could grow by utilizing metabolites of acidogenic bacteria that coexisted in the reactor. A morning feed was effective for a good growth of purple non-sulfur bacteria. A protein content of cultured bacteria was the highest when wastewater was fed in the morning. Dissolved organic carbon (DOC) removal was 94% independent of the timing control. Consequently, feeding in the morning is the optimum feed-timing control from the aspects of growth of purple non-sulfur bacteria and single-cell protein production.