Chemotherapy with low‑molecular weight compounds, despite elimination of cancer cells, entails adverse effects. To overcome this disadvantage, innovative drug delivery systems are being developed, including conjugation of macromolecular carriers with therapeutics, e.g. a nanoconjugate of hydroxyethyl starch and methotrexate (HES‑MTX). The purpose of the present study was to determine whether HES‑MTX, applied as a chemotherapeutic, is able to modulate the immune response and support the antitumor response generated by dendritic cells (DCs) used subsequently as immunotherapeutic vaccines. Therefore, MTX or HES‑MTX was administered, as sole treatment or combined with DC‑based vaccines, to MC38 colon carcinoma tumor‑bearing mice. Alterations in antitumor immune response were evaluated by multiparameter flow cytometry analyses and functional assays. The results demonstrated that the nanoconjugate possesses greater immunomodulatory potential than MTX as reflected by changes in the landscape of immune cells infiltrating the tumor and increased cytotoxicity of splenic lymphocytes. In contrast to MTX, therapy with HES‑MTX as sole treatment or combined with DC‑based vaccines, contributed to significant tumor growth inhibition. However, only treatment with HES‑MTX and DC‑based vaccines activated the systemic specific antitumor response. In conclusion, due to its immunomodulatory properties, the HES‑MTX nanoconjugate could become a potent anticancer agent used in both chemo‑ and chemoimmunotherapeutic treatment schemes.