Purpose: Atmospheric oxygen (∼20% O+) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines. Experimental Design: GL261 glioma cells and EMT6 breast carcinoma cells were grown in 5% or 20% O+. Syngeneic tumor-bearing mice were vaccinated with these tumor cell lysates mixed with CpG oligodeoxynucleotides as an adjuvant. Tumor infiltrating T cells and apoptotic GL261 cells were quantified by immunohistochemistry. Tumor-reactive immunoglobulin was detected by Western blot. Ovalbumin and gp100-derived peptides were mixed with GL261 lysates as marker antigens to detect changes in presentation of exogenous antigen on MHC class I in vitro, and in vivo following adoptive transfer of gp100-specific CD8+ T cells. Results: Mice bearing orthotopic glioma and breast carcinoma survived significantly longer when vaccinated with 5% O2 lysates. Antigen-specific CTL activation was significantly enhanced following stimulation with lysates derived from GL261 cells grown in 5% O2 versus 20% O2 through a mechanism that involved enhanced cross-presentation of exogenous antigen on MHC I. Vaccination with 5% O2 GL261 cell lysates caused a significant increase in CTL proliferation, tumoricidal function, and trafficking into brain tumor sites, whereas 20% O2 lysate vaccines predominantly evoked an antibody response. Conclusions: Tissue culture oxygen functions as an "immunologic switch" by dictating the cellular and humoral immune responses elicited by tumor cell lysates. These results have profound implications for cancer vaccines that utilize tumor cells as the source of antigen.