A B S T R A C TTwo-stage anaerobic process was studied in order to treat biodiesel wastewater. The first stage represented acidogenic reactor while the second stage was methanogenic reactor. The effect of pH, hydraulic retention time (HRT), and organic loading rate (OLR) on performance of both reactors was investigated. The optimum condition was examined using response surface methodology with the Box-Behnken Design. In the acidogenic reactor, the optimum pH, HRT, and OLR were 6.48, 16 h, and 26 g COD/(l d), respectively. High VFA production of 9.35 g/l was achieved with the low methane production. In the methanogenic reactor, the optimum pH, HRT, and OLR were 6.95, 30 h, and 6 g COD/(l d), respectively. Biogas production of 19.1 l/d was obtained with the methane content of 66%. VFA was completely consumed. In comparison, the two-stage system showed higher efficiency (COD removal, biogas production, and methane yield) than the one-stage system.