<p>Adoptive transfer of genetically or nanoparticle-engineered macrophages represents a promising cell therapy modality for treatment of solid tumor. However, the therapeutic efficacy is suboptimal without achieving a complete tumor regression, and the underlying mechanism remains elusive. Here, we discover a subpopulation of cancer cells with upregulated CD133 and programmed death-ligand 1 in mouse melanoma, resistant to the phagocytosis by the transferred macrophages. Compared to the CD133^-PD-L1^- cancer cells, the CD133⁺PD-L1⁺ cancer cells express higher transforming growth factor-β signaling molecules to foster a resistant tumor niche, that restricts the trafficking of the transferred macrophages by stiffened extracellular matrix, and inhibits their cell-killing capability by immunosuppressive factors. The CD133⁺PD-L1⁺ cancer cells exhibit tumorigenic potential. The CD133⁺PD-L1⁺ cells are further identified in the clinically metastatic melanoma. Hyperthermia reverses the resistance of CD133⁺PD-L1⁺ cancer cells through upregulating the ‘eat me’ signal calreticulin, significantly improving the efficacy of adoptive macrophage therapy. Our findings demonstrate the mechanism of resistance to adoptive macrophage therapy, and provide a de novo strategy to counteract the resistance.</p>