Abstract This study aims at developing starch based gene carriers with low cytotoxicity and high transfection efficiency. Starch molecules with molecular weight of about 50 kDa were cationically modified by spermine to obtain spermine modified starch (SMS) based gene carriers. Plasmid pAcGFP1-C1 (pDNA) was chosen as the model gene material and formed self-assembly nanocomplexes with SMS. The cytotoxicity and transfection efficiency of SMS/pDNA complexes were tested in HepG2 cell lines. Results showed that SMS/pDNA complexes formed by SMS-DS3 with the highest primary amine content (1.17 μmol mg−1) and pDNA at the weight ratio of 25 displayed the highest transfection efficiency (∼40%) with low cytotoxicity. Dynamic Light Scattering (DLS) and Small Angel X-ray Scattering (SAXS) investigation under simulated acidified endosomal environments (pH 5.0–7.4) revealed that SMS-DS3/pDNA complexes (∼180 nm) with moderately compact structures within acidic environments (i.e. no significant particle size changes, slightly shrunken shapes) showed superior transfection efficiency.