Gene delivery from biomaterials can create an environment that promotes and guides tissue formation. However, the immune response induced upon biomaterial implantation can be detrimental to tissue regeneration. Macrophages play a central role in mediating early phases of this response, and functional “polarization” of macrophages towards M1 (inflammatory) or M2 (anti-inflammatory) phenotypes may bias the local immune state at the implant site. Since gene delivery from biomaterial scaffolds can confer transgene expression in macrophages in vivo, we investigated whether transduction of macrophages with an IL-10 encoding lentivirus can (1) induce macrophage polarization toward an M2 phenotype even in an pro-inflammatory environment, and (2) prevent a shift in polarization from M2 to M1 following exposure to pro-inflammatory stimuli. IL-10 lentivirus delivery to pre-polarized M1 macrophages reduced TNF-α production 1.5-fold when compared to cells treated with either a control virus or a bolus delivery of recombinant IL-10 protein. IL-10 lentivirus delivery to naïve macrophages reduced the amount of TNF-α produced following an inflammatory challenge by 2.5-fold compared to cells treated with both the control virus and recombinant IL-10. At a mechanistic level, IL-10 lentivirus delivery mediated sustained reduction in NF-κB activation and, accordingly, reduced transcription of TNF-α. In sum, lentiviral delivery of IL-10 to macrophages represents a promising strategy for directing and sustaining macrophage polarization towards an M2 phenotype in order to promote local immune responses that facilitate tissue engineering. Biotechnol. Bioeng. 2014;111: 1210–1221. © 2014 Wiley Periodicals, Inc.