Dysregulation of apoptosis is associated with the development of human cancer and resistance to anti-cancer therapy. The ultimate goal of cancer treatment is to selectively induce cancer cell death and to overcome drug resistance. A deeper understanding of how chemotherapy affects tumor cell death is needed in order to develop strategically designed anti-cancer agents. An athymic mouse xenograft tumor system was established utilizing genetically defined cells that are deficient in apoptosis to examine the involvement of multiple forms of cell death induced by a DNA alkylating agent commonly used in chemotherapy. In response to DNA alkylating damage, although apoptosis facilitates a more rapid tumor regression, it is dispensable for complete tumor regression as other forms of cell death such as sporadic necrosis, senescence, autophagy, and mitotic catastrophe are activated. Of these, sporadic necrosis plays a fundamental role in tumor clearance by stimulating the innate immune response in a manner that is dependent upon the high mobility group box 1 (HMGB1) protein. HMGB1 is released from necrotic cells and once outside the cell, it acts as a damage-associated molecular pattern (DAMP) molecule to alert the host of damage by triggering immune responses. Interestingly, while DNA alkylating therapy leads to complete tumor regression in this mouse model, tumors deficient in HMGB1 fail to fully regress. The HMGB1-deficient tumors have an attenuated ability to recruit innate immune cells including macrophages, neutrophils, and natural killer (NK) cells into the treated tumor tissue. Cytokine array analysis reveals that while DNA alkylating treatment leads to suppression of pro-tumor cytokines such as IL-4, IL-10, and IL-13, loss of HMGB1 leads to elevated levels of these cytokines upon treatment. Suppression of innate immunity and HMGB1 using neutralizing antibodies leads to a failure in tumor regression. Taken together, these results indicate that DNA alkylating therapy can induce multiple forms of cell death and extracellular release of cellular contents such as HMGB1, which in turn leads to the activation of innate immunity and tumor regression.