The performance of traditional heterologous protein production by Pichia pastoris with methanol induction at 30 ℃ is poor, characterized by low ATP regeneration rate and weak operation stability. A low temperature induction strategy at 20 ℃ was thus adopted for efficient porcine interferon-α production in a 10 L fermentor. With the strategy, maximal methanol tolerance level could reach about 40 g/L to effectively deal with methanol concentration variations, so that the complicated on-line methanol measurement system could be eliminated. Moreover, metabolic analysis based on multiple state-variables measurements indicated that pIFN-α antiviral activity enhancement profited from the formation of an efficient ATP regeneration system at 20 ℃induction. Compared to the induction strategy at 30 ℃, the proposed strategy increased the ATP regeneration rate by 49-66%, the maximal pIFN-α antiviral activity was enhanced about 20-fold and reached a higher level of 1.5×106 IU/mL.