Fed-batch fermentation of a methanol utilization plus (Mut(+)) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut(+) Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate ( micro ) and specific glycerol/methanol consumption rate ( nu(gly), nu(MeOH)) were established. A mixed feeding strategy with desired micro (gly)/ micro (MeOH) =1, 2, 3, 4 (desired micro (MeOH) set at 0.015 h(-1)) was employed to study growth interactions and their effect on production. The results show that the optimal desired micro (gly)/ micro (MeOH) is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3 mg/g wet cells.