The proteome of Pichia pastoris fermentation culture medium was studied to reveal protein identities and to enhance recombinant protein purification and secretion in P. pastoris. Protein samples from the culture media and 2D gel spots were subjected to ESI-MS/MS analysis and search against public fungi genome database. Homology MS-BLAST search was also performed to provide information of single peptide hit and unidentified gel spots. A total of thirty-three proteins were identified in different fermentation cultures, including before and after methanol induction, different temperatures and in recombinant protein expression cultures. In a fed-batch fermentation, cultivation at 30 °C showed higher protein concentration and more protein species than 25°C cultures, suggesting cultures at 30°C exhibit a greater challenge for recombinant protein purification. Most of the identified proteins, from medium solutions and 2D gel spots, have low isoelectric points (pI<6) with both naturally secreted proteins and intracellular proteins. Different glucanases, including EXG, Gas1p and some cell wall proteins, comprised the majority of the secreted proteins. Intracellular proteins such as alcohol oxidase and peroxiredoxin were also found in the post-methanol induction samples. This study provides the real-time presence of different proteins in the fermentation culture media and suggests the potential biotechnology applications based on the discovery of this proteome.