Macrophages play a critical role in mediating not only normal tissue healing, but also the host reaction against biomaterial scaffolds. There is increasing interest in regenerative medicine to combine mesenchymal stromal/stem cells (MSCs) with biomaterial scaffolds to modulate inflammatory response while restoring tissue architecture. The objective of the current study was to investigate the interaction between MSCs (derived from bone marrow, adipose or vocal fold tissue) encapsulated in hyaluronan‐based hydrogel and differentiating macrophages as measured by extracellular matrix (ECM) gene expression and cytokine, chemokine, and growth factor concentrations. Gene expression was analyzed using real‐time polymerase chain reaction from MSCs embedded in Carbylan‐GSX after 7 days of coculture with or without CD14+ cells. Protein concentrations were measured using a Bio‐plex assay from cell culture supernatants on days 3 and 7 for all conditions. Following 7 days, we identified upregulation of collagen‐I, collagen‐III, procollagen, and matrix metalloproteinase‐9 genes compared to control conditions. We demonstrate increased concentrations of immunoregulatory cytokines interleukin (IL)‐1β, tumor necrosis factor‐α, macrophage inflammatory protein‐1α, IFN‐γ, IL‐12, and IL‐10 and remodeling growth factors (vascular endothelial growth factor, hepatocyte growth factor) in MSC‐3D constructs cocultured with macrophages compared to control conditions, with some temporal variation. Our results indicate an alteration of expression of ECM proteins important to tissue regeneration and cytokines critical to the inflammatory cascade when 3D constructs were cultured with differentiating macrophages. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 890–902, 2014.