Gene therapy has great potential in disease prevention and treatment. The purpose of the proposed research was to advance the development of safe and effective nonviral polymeric vectors for targeted gene therapy and DNA vaccine delivery. A series of hydrophobically modified chitosan derivatives with increasing degrees of chain length, substitution, and hydrophobicity was synthesized via carbodiimide mediated coupling reaction. The chemical structure of the polymers was determined using proton nuclear magnetic resonance (1H NMR), fourier transform infrared (FTIR) spectroscopy, and elemental analysis. These polymers form micellar structures in aqueous environment and effectively condense plasmid DNA (pDNA) into nanoscale polyplexes. The acyl chain length, degree of substitution, and hydrophobicity of the substituent had great impact on the particle size, pDNA binding strength, in vitro pDNA release profile, cellular uptake, and in vitro gene transfection efficiency of the polymer/pDNA polyplexes. The hexanoic acid grafted chitosan [NAC-6(15)] and L-phenylalanine grafted chitosan (AGC-F) with a 15% degree of amino substitution demonstrated significantly (p < 0.05) higher gene transfection in HEK 293 cells, and their transfection efficiency surpassed the transfection capacity of FuGENE HD. The NAC-6(15) and AGC-F polymers were mannosylated to provide selective antigen presenting cell (APC) targeting, thereby facilitating cellular uptake to ultimately improve the overall immune response to the DNA vaccine. The chemical composition of the mannosylated copolymers was analyzed by 1H NMR spectroscopy. These polymers efficiently condense pDNA into nanosized polyplexes with net positive surface charges. The resultant polyplexes demonstrated excellent protection of the condensed pDNA from enzymatic degradation by deoxyribonuclease (DNase). The synthesized mannosylated polymers exhibited 7-fold greater cellular uptake than chitosan in RAW 264.7 cells, which express mannose receptors, mainly via the receptor-mediated endocytosis without affecting biocompatibility. The in vitro transfection efficiencies of mannosylated polymer/pDNA polyplexes were significantly (p < 0.05) higher than other polymers and FuGENE HD. An in vivo study in Balb/c mice using hepatitis B surface antigen encoding pDNA as a model DNA vaccine reflected good efficacy and biocompatibility of the delivery system. Therefore, hydrophobically modified mannosylated chitosan derivatives have the potential to be a safe and efficient APC targeting gene carrier for DNA vaccine delivery.