Tumor-associated macrophages (TAM) have important roles in cancer promotion, but the signalling behind the formation of protumoral TAM remains understudied. Here, by single-cell RNA sequencing, we revealed that the pattern recognition receptor Mincle was highly expressed in TAM of patients and significantly associated with the mortality in non-small cell lung cancer patients. Cancer cells markedly induced Mincle expression in bone marrow-derived macrophage (BMDM), thus promoting cancer progression in invasive lung carcinoma LLC and melanoma B16F10 in vivo and in vitro. Mincle was predominately expressed in the M2-like TAM in NSCLC and LLC-tumors and silencing of Mincle unexpectedly promoted M1-like phenotypes in vitro. Mechanistically, we discovered a novel Mincle/Syk/NF-κB signalling pathway in TAM needed for executing their TLR4-independent protumoral activities. Adoptive transfer of Mincle-silenced BMDM significantly suppressed TAM-driven cancer progression in the LLC-bearing NOD/SCID mice. By modifying our well-established ultrasound microbubble-mediated gene transfer protocol, we demonstrated that tumor-specific silencing of Mincle effectively blocked Mincle/Syk/NF-κB signalling, therefore inhibiting the TAM-driven cancer progression in the syngernic mouse cancer models. Thus, our findings highlighted the function of Mincle as a novel immunotherapeutic target for cancer via targeting the Mincle/Syk/NF-κB circuit in TAM. Copyright ©2020, American Association for Cancer Research.