In production of porcine interferon a (pIFN-alpha) by Pichia pastoris, improper glycerol feeding strategy leads to ethanol accumulation in the last stage of growth phase. In the present study, taking two runs with low ethanol accumulation under 2 g/L as control group, effects of long-term (>4 h) and instantaneous high ethanol concentration (> 10 g/L) on pIFN-alpha production, and activities of key enzymes in carbon metabolism were discussed. As a result, compared with control group, pIFN-a expression level was decreased about 4 similar to 12 folds under long-term high ethanol concentration, from the level above 3 g/L to the level under 1 g/L; pIFN-alpha expression level was decreased about 8 folds under instantaneous high ethanol concentration, reaching to the low level of 0.42 g/L. The low production of pIFN-alpha was caused by the severe inhibitory effect of ethanol on these enzymes.