In the majority of human tumors, downregulation of major histocompatibility complex class I (MHC-I) expression contributes to the escape from the host immune system and resistance to immunotherapy. Relevant animal models are therefore needed to enhance the efficacy of cancer immunotherapy. As loss of β-2 microglobulin expression results in irreversible downregulation of surface MHC-I molecules in various human tumors, the β-2 microglobulin gene (B2m) was deactivated in a mouse oncogenic TC-1 cell line and a TC-1/dB2m cell line that was negative for surface MHC-I expression was derived. Following stimulation with interferon γ, MHC-I heavy chains, particularly the H-2D b molecules, were found to be expressed at low levels on the cell surface, but without β-2 microglobulin. B2m deactivation in TC-1/dB2m cells led to reduced proliferation and tumor growth. These cells were insensitive to DNA vaccination and only weakly responsive to combined immunotherapy with a DNA vaccine and the ODN1826 adjuvant. In vivo depletion demonstrated that NK1.1 + cells were involved in both reduced tumor growth and an antitumor effect of immunotherapy. The number of immune cells infiltrating TC-1/dB2m-induced tumors was comparable with that in tumors developing from TC-1/A9 cells characterized by reversible MHC-I downregulation. However, the composition of the cell infiltrate was different and, most importantly, infiltration with immune cells was not increased in TC-1/dB2m tumors after immunotherapy. Therefore, the TC-1/dB2m cell line represents a clinically relevant tumor model that may be used for enhancement of cancer immunotherapy.