Macrophages play a fundamental role in innate immunity by phagocytosing and eliminating invading pathogens upon their pro-inflammatory activation. Conversely, anti-inflammatory macrophages promote tissue regeneration and extracellular matrix (ECM) remodeling. While macrophage activation is a well-recognised modulator of tissue homeostasis, the influence of their local tissue microenvironment is not understood. In this study, we set out to examine the effects of ECM protein composition and tissue stiffness on macrophage activation. Bone marrow derived-macrophages (BMDM) or RAW264.7 murine macrophage cell lines were cultured in 3D by suspension within collagen gels. Macrophages cultured in 3D were compared to 2D controls (traditional plastic tissue-culture plates either with no ECM or a thin collagen monolayer), and a 2D cushion model (cells cultured as a monolayer on top of a pre-set collagen gel). Viability of BMDMs grown in 2D was ~80% after 6 days culture with media replacement every 2 days. By contrast, BMDM viability in the 2D cushion model was to ~75%, and only ~10% in the 3D model after 6 days, despite nutrient replenishment. By contrast, RAW264.7 macrophages exhibited increased viability in 3D compared to BMDM, with ~60% live cells after 6 days culture. Moreover, a human prostate cancer cell line (PC3*) exhibited normal growth and viability in 3D, suggesting BMDM are particularly sensitive to their culture environment. To determine whether 3D culture influenced the ability of macrophages to clear pathogens, we next infected BMDM with the protozoan parasite Leishmania mexicana prior to seeding within collagen gels. Strikingly, infected BMDM in 3D showed an increased viability to ~60% after 6 days of culture, compared to ~10% in uninfected 3D counterparts. All together, we show that culturing macrophages in 3D has an effect on their biology and may ultimately facilitate more accurate drug screening or the development of new therapeutics.