Tumor-associated macrophages (TAMs) support tumor growth by suppressing the activity of tumor-infiltrating T cells. Consistently, TAMs are considered a major limitation for the efficacy of cancer immunotherapy. However, the molecular reason behind the acquisition of an immunosuppressive TAM phenotype is not fully clarified. During tumor growth, the extracellular matrix (ECM) is degraded and substituted with a tumor-specific collagen-rich ECM. The collagen density of this tumor ECM has been associated with poor patient prognosis but the reason for this is not well understood. In this study, we investigated whether the collagen density could modulate the immunosuppressive activity of TAMs. The murine macrophage cell line RAW 264.7 was three-dimensionally cultured in collagen matrices of low and high collagen densities mimicking healthy and tumor tissue, respectively. Collagen density did not affect proliferation or viability of the macrophages. However, whole-transcriptome analysis revealed a striking response to the surrounding collagen density, including the regulation of immune regulatory genes and genes encoding chemokines. These transcriptional changes were shown to be similar in murine bone marrow-derived macrophages and TAMs isolated from murine tumors. Strikingly, coculture assays with primary T cells showed that macrophages cultured in high-density collagen were less efficient at attracting cytotoxic T cells and capable of inhibiting T cell proliferation more than macrophages cultured in low-density collagen. Our study demonstrates that a high collagen density can instruct macrophages to acquire an immunosuppressive phenotype. This mechanism could reduce the efficacy of immunotherapy and explain the link between high collagen density and poor prognosis.