Macrophages are present within all tissues where they exhibit a wide variety of functions, ranging from the phagocytosis and elimination of intracellular pathogens to homeostatic roles in organ function and development. Developing these varied, highly specialised functions is dependent on the macrophage sensing and correctly integrating cues from their complex, three-dimensional (3D) tissue niche. Yet, in the laboratory, macrophages are routinely cultured in vitro on stiff, flat, plastic cultureware. We set out to establish a 3D model that better mimics in vivo tissue niches and examine the influence of this microenvironment on the activation and metabolism of macrophages. Bone marrow derived-macrophages (BMDM) or RAW264.7 murine macrophage cell lines were suspended in 3D within collagen gels. These cells were compared to 2D controls (traditional plastic tissue-culture plates coated with a thin extracellular matrix (ECM) protein monolayer), and a 2D cushion model (cells cultured as a monolayer on top of a pre-set collagen gel). After 3 and 6 days in culture, the viability of BMDM cultured in 3D was significantly lower than 2D controls and RAW264.7 cells in 3D. We screened a variety of additional ECM proteins and identified that integration of vitronectin into 3D cultures restored BMDM viability in 3D. We next set out to examine the effect of 3D culture on macrophage activation and metabolism. In traditional 2D conditions, BMDM stimulated with pro-inflammatory cues (lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)) upregulate the signature gene Nos2, concomitant with increased glycolytic flux and absent oxidative respiration, while stimulation with anti-inflammatory cues (IL-4 and IL-13) induces Mrc1 expression and increases oxidative respiration. In 3D, BMDM activated to both pro- and anti-inflammatory phenotypes had significantly increased mitochondrial and glycolytic metabolism than 2D controls. This was associated with an increased expression of anti-inflammatory genes (Mrc1, Arg1) and reduced expression of Nos2 and nitric oxide production. Taken together, our data shows that 3D culture influences macrophage activation into pro- and anti-inflammatory phenotypes and alters the cellular metabolism of macrophages, overall inducing them to be more energetic. This project is the first step on the road to better modelling macrophage biology in tissue microenvironments.