Response surface methodology (RSM) is utilized to analyze and optimize the experimental condition of V(Ⅴ) removal in single chamber microbial fuel cell (MFC). The influences of initial vanadium concentration, COD and electrolyte conductivity on V(Ⅴ) removal efficiencies are investigated when sodium acetate is adopted as carbon source in single chamber MFC. The results of RSM with three factors at three levels reveal that the initial vanadium concentration has the most significant effect on V(Ⅴ) removal efficiencies, followed by the COD, whereas the influence of the electrolyte conductivity is minimal. The optimal experimental condition obtained by RSM is initial V(Ⅴ) concentration of 75.44 mg/L, COD of 1007.48 mg/L and electrolyte conductivity of 11.98 mS/cm, in which condition the theoretical maximum V(Ⅴ) removal efficiency of 80.31% can be achieved. Verification test is also conducted and the results further confirm the reliability of the optimization. The results of the present study will promote the application of MFC in the remediation of vanadium contaminated groundwater.