λ-Carrageenan is a seaweed polysaccharide which has been generally used as proinflammatory agent in the basic research, however, how the immunomodulating activity of λ-carrageenan affects tumor microenvironment remains unknown. In this study, we found that intratumoral injection of λ-carrageenan could inhibit tumor growth in B16-F10 and 4T1 bearing mice and enhance tumor immune response by increasing the number of tumor-infiltrating M1 macrophages, DCs and more activated CD4(+)CD8(+) T lymphocytes in spleen. In addition, λ-carrageenan could enhance the secretion of IL17A in spleen and significantly increase the level of TNF-α in tumor, most of which was secreted by infiltrating macrophages. Moreover, λ-carrageenan exhibited an efficient adjuvant effect in OVA-based preventative and therapeutic vaccine for cancer treatment, which significantly enhanced the production of anti-OVA antibody. The toxicity analysis suggested that λ-carrageenan was with a good safety profile. Thus, λ-carrageenan might be used both as a potent antitumor agent and an efficient adjuvant in cancer immunotherapy.