Abstract: Although high lignocellulose content decreases acidification rate of anaerobic fermentation of cattle manure, acidification phase is critical in anaerobic fermentation of cattle manure. Cattle manure acidification conditions were investigated with the aim of optimizing methane production in a two-phased anaerobic process of cattle manure. The effects of acidification time, stirring frequency, feed solution concentration and urea addition for cattle manure methane production were studied in a two-phased anaerobic fermentation at 35 ℃. Single factor experiments showed that 96 h of acidification, 3 times in every 24 h (60 r·min^-1, 1 min·time^-1) of stirring frequency, 8.0% feed solution concentration and 1.28 g·L^-1 urea addition were the optimum conditions. Based on single factor experiments, a series of central composite design experiments were conducted for optimum synthesis. Response surface methodology (RSM) with central composite design (CCD) was used to explore optimum conditions for methane production. The effects of three variables (acidification time, feed solution concentration and urea addition) were subsequently appraised. A regression model for methane production under the acidification conditions was established for analysis of interactions and significance in accordance with central composite design. Results showed that methane production was greatly influenced by feed solution concentration, urea addition and acidification time under at 35 ℃ and 3 times per every 24 h (60 r·min^-1, 1 min·time^-1) stirring frequency. The optimum acidification conditions occurred at the acidification time of 93.7 h, feed solution concentration of 8.3% and urea addition of 1.26 g·L^-1. Under the optimum condition, methane content and total production increase by 14.3% and 44.7%. Also the removal rates of volatile solid (VS) and chemical oxygen demand (COD) increased by 41.8% and 33.9%, respectively. However, the contents of cellulose, hemicellulose and lignin were not affected by cattle manure acidification. The findings indicated that cattle manure acidification improved methane production, methane content, and VS and COD removal rates.