Chitosan has emerged as a promising polysaccharide for gene/siRNA delivery. However, additional works will be required to modify chitosan nanoparticles. In the present study, chitosan nanoparticles were well modified to introduce anti-HIV siRNA into two mammalian cell lines, macrophage RAW 264.7 and HEK293. We first generated two stable cell lines expressing HIV-1 Tat, and then designed and generated an efficient anti-tat siRNA. The nanoparticles were prepared by using different concentrations of chitosan, polyethylenimine (PEI) and carboxymethyl dextran (CMD) in various formulations and then their physicochemical and biological properties were investigated. The results demonstrated that the combination of chitosan with both CMD and PEI significantly improved both cell viability and siRNA delivery. The modified chitosan nanoparticles (ChNPs) at the N:P ratio of 50 were approximately uniform spheres with sizes ranging from 100 to 150 nm and a positive zeta potential of about +22 mV. In both cell types, the nanoparticles noticeably increased siRNA delivery efficiency with no significant cytotoxicity or apoptosis-inducing effects compared to the control cells. In addition, the nanoparticles significantly reduced the RNA and protein expression of HIV-1 tat in both stable cells. These data show that the nanoparticle formulation could potentially be used in gene therapy, especially against HIV infection. Display omitted •Generation of two stable cell lines, macrophage RAW 264.7 and HEK293, expressing HIV-1 Tat.•Designing specific siRNAs targeting HIV-1 tat gene.•Synthesis of chitosan nanoparticles and modification by polyethylenimine (PEI) and carboxymethyl dextran (CMD).•High cell viability and siRNA delivery efficiency by adjusting the molar ratio of precursors (siRNA, chitosan, CMD and PEI).•Effectively suppression of HIV-1 Tat by the siRNA-loaded nanoparticles in stable cells.