Of the non-viral vectors, a cationic polymer like PEI is an attractive candidate which however, has been negatively impacted due to its marked toxicity. An anionic sugar polymer gelan gum (GG) has been introduced into PEI system to increase transfection efficiency with minimal toxicity. We showed that one of the synthesized (GP1-GP6) GG-PEI nanocomposites (NCs), GP3, exhibited negligible toxicity in in vitro (primary keratinocytes, HEK293, HeLa and HepG2 cells) and in vivo (Drosophila melanogaster) as compared to PEI or lipofectamin. GP3-pDNA complex was found to be transfected efficiently in the above cells as confirmed by FACS analysis (72.0 + 5.5%) while lipofectamine showed only 12.4 + 3.5% efficiency. GP3 mediated GFP specific siRNA delivery resulted in the knockdown of the GFP expression by approximately 77% and JNK (60%). In vivo gene expression studies in mice revealed reporter gene expression in spleen. The study demonstrates that GG blended PEI NCs hold promise for future applications in gene delivery both in vitro and in vivo.