Abstract: Total protein extraction of Streptomyces luteogriseus in different fermentation times was analyzed in order to discover the enzymes associated with Maituolaimysin biosynthesis, make the biosynthesis pathway clear, and enhance its production. Two-dimensional gel electrophoresis analysis showed that 8 proteins were up-expressed, 5 were down-expressed, 6 were new-expressed and 6 disappeared in the protein extraction of 48 h compared with that of 36 h. Fifteen of the different expression proteins were analyzed by Matrix-Assisted Laser Desorption Ionization(MALDI)Time-of-Flight(TOF) mass spectrum and 4 of them were identified. Among the 4 identified proteins, one was involved in protein biosynthesis; one was associated with Streptomyces differentiation; one was a key enzyme in TCA circle and another one was a hypothetical protein whose function was unknown.Investigation of the function of these identified proteins suggested that they all had direct or indirect relations to Maituolaimysin biosynthesis, and especially, the protein Clp2 could accelerate the differentiation of Streptomyces, which would directly result in Maituolaimysin biosynthesis.