Rhizopus lipases have been successfully expressed in Pichia pastors and different fermentation strategies have been investigated. However, there is no sufficient study on the effects of methanol concentration on the production of Rhizopus lipases in P. pastors. In this study, the lipase from Rhizopus chinensis CCTCC M20102 was expressed under different fed-batch fermentation conditions at methanol concentrations ranging from 0.5 to 3.5 g/L. The lipase activity, stability, and productivities were analyzed. The optimum methanol concentration was 1 g/L, with the highest lipase activity of 2,130 U/mL, without degradation. Additional information was obtained from the analysis of methanol consumption and production rates. The results also suggested that the cell concentration at the end of the glycerol fed-batch phase was very important for cell viability and protease activity.