In this study, cationic nanoparticles self-assembled from the amphiphilic copolymer poly(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate) (P(MDS-co-CES) were synthesized and used to deliver Bcl-2 targeted siRNA into HepG2, HeLa and MDA-MB-231 cell lines, and downregulate Bcl-2 mRNA expression levels. Confocal microscopic studies show that the nanoparticles were able to complex with siRNA and deliver it inside the cells efficiently, but siRNA was easily dissociated from the complexes in the cytoplasm for its biological functions. Bcl-2 mRNA expression levels as low as 10% were achieved after treatment with nanoparticle/siRNA complexes. The downregulation efficiency of Bcl-2 mRNA level was similar to that mediated by Lipofectamine but higher than that induced by PEI. PEG was also conjugated to siRNA via a cleavable disulfide bond, and nanoparticle/siRNA-PEG complexes showed no significant protein adsorption as compared with 26 and 17% for blank nanoparticles and nanoparticle/siRNA complexes, respectively. The presence of serum caused slight aggregation of nanoparticle/siRNA or nanoparticle/siRNA-PEG complexes. However, the size of the complexes was still below 250 nm after being incubated in PBS containing 10% serum for 4 h. On the other hand, PEGylated siRNA delivered by the nanoparticles downregulated Bcl-2 mRNA expression level in the cells as efficiently as unmodified siRNA. Bcl-2 protein was also downregulated efficiently by nanoparticle/siRNA complexes in all cell lines tested. The downregulation of Bcl-2 mRNA or Bcl-2 protein did not show significant cell death in the tested siRNA and polymer concentration range. However, the delivery of siRNA sensitized HeLa cells to paclitaxel treatment, yielding significant improvement over the untreated cells (p < 0.05). These cationic nanoparticles may be potentially employed to downregulate Bcl-2 expression and sensitize cancer cells to anticancer drugs for more efficient chemotherapy.