Penetration and distribution of drug through the avascular regions of human solid tumors after extravasation are crucial concerns for antitumor efficacy. To address this issue, an in vitro solid tumor model of multicellular layers (MCLs) of human colorectal cancer cells (DLD-1) was established. In an attempt to deliver Dicer substrate small interfering RNA (DsiRNA), chitosan (CS) nanoparticles have been developed for targeting vascular endothelial growth factor (VEGF) gene for tumor growth inhibition. The DsiRNA-CS nanoparticles prepared by ionic gelation method had provided maximal protection of DsiRNA in full human serum up to 48 h incubation. RT-PCR studies revealed significant concentration- and time-dependent knock-down of VEGF mRNA and its product due to uniform penetration of DsiRNA-CS nanoparticles throughout MCLs. Taken together, this study also demonstrated that DsiRNA-CS nanoparticles could effectively knock down VEGF gene as therapeutic target in monolayer culture or in solid tumor model for potential treatment of human colorectal carcinoma.