Abstract: There exists a transition phase from growth phase with glycerol as carbon source to expression phase with methanol as carbon source when Pichia pastoris was used as heterogous protein expression system. The key enzymes of main metabolism pathways at the transition phase were analyzed in this (paper.) It was shown that alcohol oxidase(AOX2) activity was not detected at 0 h and 3 h when induced with methanol, whereas its activity sharply increased to 0.05 U at 4 h after induction, and AOX2 activity (increased) slowly afterwards. During transition phase formaldehyde dehydrogenase(EC 1.2.1.1 FAD) and glucose-6-phosphate dehydrogenase(EC 1.1.1.49, G-6-P) increased 6.1 times and 2.5 times, while pyruvate dehydrogenase complex(PDC) and isocitrate dehydrogenase(EC 1.1.1.42,IDE) decreased by 70.6% and 83.6%, respectively compared with the activities at the end of growth phase. Therefore, we inferred that the main methanol metabolic pathway was that methanol was completely oxidized to exhaust CO_2 and that metabolic activities of glycolysis pathway and tricarboxylic acids cycle were attenuated when carbon source was changed from glycerol to methanol.