Cancer is the second leading cause of death worldwide, the deaths are projected to continue rising, with an estimated 12 million deaths in 2030. The aim of the present investigation is to prepare and compare the uncoated (U-CH NP) and eudragit S 100-coated (E-U-CH NP) chitosan nanoparticles encapsulating a caspase 3 activator (UCN 01), by ionic gelation method. The prepared formulations were studied for various parameters like particles size, zeta potential, transmission electron microscopy, atomic force microscopy, in vitro release study, ex vivo study using Caco 2 colon cancer cell line, and in vivo studies. The particle size and zeta potential of developed formulation was found to be particle size of 168 ± 3.7 nm and +35.8 ± 3.7 for U-CH NP and 265 ± 4.1 nm and +22.3 ± 1.1 for E-U-CH NP. TEM and AFM images revealed that U-CH NPs were round in shape and smoother at surface as compared to E-U-CH NP which have irregular surface due to coating. The E-U-CH NP showed better in vitro release than uncoated formulation in SCF (pH 6.8) than in SGF (pH 1.2). The cytotoxicity was performed by MTT assay. U-CH NP showed enhanced cytotoxicity as compared to blank (without drug) formulation. There was an increase in caspase 3 activity of U-CH NP as compared to UCN 01 alone. E-U-CH NP showed better tumor regression ability than U-CH NP. The results of plasma profile and tumor regression study demonstrated that E-U-CH NP has continuous release profile of UCN 01 and comprehensive residence time. Thus, it is better acceptable than free UCN 01 and may be a potential delivery system for the targeting and treatment of colon cancer.