Myeloid-derived suppressive cells (MDSC) inhibit antitumor immunity and confer a survival advantage for tumor evasion. Tumor cells also support MDSC expansion and recruitment by secreting multiple growth factors and cytokines, but the mechanisms by which tumors affect MDSC function are not completely understood. Here, we found that the neuronal guidance protein netrin-1 was selectively secreted by MC38 murine colon cancer cells, which could enhance the immunosuppressive activity of MDSCs. MDSCs predominantly expressed one type of netrin-1 receptor, adenosine receptor 2B (A2BR). Netrin-1 interacted with A2BR on MDSCs to activate the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway, which ultimately increased CREB phosphorylation in MDSCs. Furthermore, netrin-1 knockdown in tumor cells inhibited the immunosuppressive activity of MDSCs and restored antitumor immunity in MC38 tumor xenograft mice. Intriguingly, high netrin-1 in the plasma correlated with MDSCs in patients with colorectal cancer. In conclusion, netrin-1 significantly enhanced the immunosuppressive function of MDSCs through A2BR on MDSCs, thus promoting the development of tumors. These findings highlight that netrin-1 may regulate the abnormal immune response in colorectal cancer and may become a potential target for immunotherapy.