Nonviral gene delivery systems have a number of limitations including low transfection efficiency, specificity, and cytotoxicity, especially when the target cells are macrophages. To address these issues, the hypothesis tested in this study was that mannose functionalized nanohybrids composed of synthetic and natural polymers will improve transfection efficiency, cell viability, and cell specificity in macrophages. Robust nanohybrids were designed from hyaluronic acid (HA) and branched polyethyleneimine (bPEI) using carbodiimide chemistry. The reaction product, i.e., branched polyethyleneimine-hyaluronic acid (bPEI-HA) copolymer was subsequently functionalized with mannose at the terminal end of the copolymer to obtain mannosylated-bPEI-HA (Man-bPEI-HA) copolymer. UV spectroscopy and gel retardation studies confirmed the formation of polyplexes at polymer to DNA weight ratio ≥ 2. Alamar Blue and MTT assay revealed that the cytotoxicity of the developed nanohybrids were significantly (P < 0.05) lower than that of unmodified bPEI. Mannose functionalization of these nanohybrids showed specificity for both murine and human macrophage-like cell lines RAW 264.7 and human acute monocytic leukemia cell line (THP1), respectively, with a significant level (P < 0.05) of expression of gaussia luciferase (GLuc) and green fluorescent reporter plasmids. Internalization studies indicate that a mannose mediated endocytic pathway is responsible for this higher transfection rate. These results suggest that hyaluronan-based mannosylated nanohybrids could be used as efficient carriers for targeted gene delivery to macrophages.