Here, we designed a new nano-sized siRNA carrier system composed of biocompatible/biodegradable glycol chitosan polymer (GC) and strongly positively charged polyethylenimine (PEI) polymers. In order to make a stable and tumor-homing nano-sized carrier, each polymer was modified with hydrophobic 5β-cholanic acid, and they were simply mixed to form self-assembled GC–PEI nanoparticles (GC–PEI NPs), due to the strong hydrophobic interactions of 5β-cholanic acids in the polymers. The freshly prepared GC–PEI NPs showed a stable nanoparticle structure (350 nm) and they presented a strongly positive-charged surface (ζ potential = 23.8) that is enough to complex tightly with negatively charged RFP-siRNAs, designed for inhibiting red fluorescent protein (RFP) expression. The siRNA encapsulated nanoparticles (siRNA–GC–PEI NPs) formed more compact and stable nanoparticle structures (250 nm) at 1: 5 weight ratio of siRNA to GC–PEI nanoparticles. In vitro RFP expressing B16F10 tumor cell (RFP/B16F10) culture system, the siRNA–GC–PEI NPs presented a rapid time-dependent cellular uptake profile within 1 h. Moreover, the internalized siRNA–GC–PEI NPs lead to specific mRNA breaks down. Furthermore, our new formulation of siRNA–GC–PEI NPs presented a significant inhibition of RFP gene expression of RFP/B16F10-bearing mice, due to their higher tumor-targeting ability. These results revealed the promising potential of GC–PEI NPs as a stable and effective nano-sized siRNA delivery system for cancer treatment.