Although immunotherapy has shown promising results in the treatment of cancer, clinical studies assessing immunologic approaches in patients with advanced cancer will seldom be conducted in the absence of conventional treatment strategies such as chemotherapy. Here we investigate the combination of chemotherapy with CpG oligonucleotide and dendritic cell-based immunotherapy in the C26 mouse model of colon carcinoma. The coinjection of antigen-pulsed, mature dendritic cells and CpG oligonucleotides together with a peritumoral injection of CpG oligonucleotides elicits a CD8 T-cell response resulting in tumor rejection and long-term protection in the C26 model. Tumor-bearing mice were treated weekly for 4 weeks by this immunotherapy protocol, by 5-fluorouracil plus leucovorin or irinotecan, or by the combination of immunotherapy and chemotherapy. We observed that immunotherapy was more effective in reducing tumor growth and increasing survival than 5-fluorouracil or irinotecan. Immunotherapy was well tolerated, whereas therapeutic doses of 5-fluorouracil or irinotecan were associated with dose-limiting toxicity. Furthermore, the efficacy of immunotherapy combined with either 5-fluorouracil or irinotecan was similar to that of immunotherapy alone. Addition of immunotherapy to either 5-fluorouracil or irinotecan treatment strongly decreased the toxicity of chemotherapy. Immunotherapy both with and without chemotherapy generated a memory immune response, leading to tumor rejection in mice rechallenged with C26 tumor cells up to several months after treatment. In summary, immunotherapy with a combination of dendritic cells and CpG oligonucleotides is superior to chemotherapy in the C26 tumor model. This immunotherapy protocol can be combined with current chemotherapy agents with no loss in therapeutic activity. © 2005 Wiley-Liss, Inc.