Gene therapy has drawn significant interest in the past two decades since it provides a promising strategy to treat both genetic disorders and acquired diseases. However, the transfer of gene therapy to clinical applications is troubled with many difficulties, since many current systems are of toxicity, low transfection efficiency and low biodegradability. To address these challenges, we developed a dextran-peptide hybrid system as a safe and efficient vector for gene therapy and investigated the structure-function-cytotoxicity relationship of this dextran-peptide hybrid system. Dextrans (Dex10, Dex20, and Dex70) with different molecular weights (10, 20 and 70 kDa) were conjugated with a cationic peptide, R5H5, at various degrees of substitution. Gene expression and cytotoxicity mediated by this delivery system were evaluated against SKOV-3 human ovarian carcinoma cells and compared to 25 kDa branched poly(ethylenimine) (PEI). The results showed that Dex10-R5H5 and Dex20-R5H5 hybrids derived from low molecular weight dextrans induced higher gene expression and lower cytotoxicity than Dex70-R5H5 hybrid from higher molecular weight dextran. The best performance on gene expression was achieved by Dex10-R5H5 at 40% substitution of R5H5, which induced greater gene expression than PEI at a low N/P ratio of S. Dex10-R5H5/DNA complexes at 40% substitution of R5H5 also showed much higher cell viability (93%) than PEI/DNA (66%) at the same N/P ratio. These results indicate that the Dex-R5H5 hybrid with the low molecular weight of dextran and the high degree of substitution of R5H5 is a very promising material for safe and efficient gene therapy.