Selenium (Se), a nutrient trace mineral, plays important roles in optimizing human health. Chitosan is an effective, natural-oriented material for synthesizing nanopolymers, with preferable properties such as biocompatibility, biodegradation and resistance to certain enzymes. In this study, encapsulated Na2SeO3 and methylseleninic acid (MSeA) with low and medium molecular weight chitosan were used to determine the efficacy of Se in mitigating tumorigenesis. We applied Se compounds, which is from sub-lethal to lethal dose, to colon cancer cell line HCT-116 and normal fibroblasts cell line MRC-5. Analysis of cellular selenium content demonstrated that: 1) Na2SeO3, but not MSeA, treatment resulted in a greater Se retention in HCT-116 than in MRC-5 cells, 2) chitosan encapsulation enhanced Se contents in cells treated with the various Se preparations. Cell survival analysis showed that chitosan encapsulation protected HCT-116 and MRC-5 cells from Na2SeO3 or MSeA induced toxicity. Moreover, this beneficial effect was greater in MRC-5 cells. MSeA encapsulated with chitosan induced phosphorylated ATM Ser-1981 formation in MRC-5 and HCT-116 cells to a less extent as compared to MSeA alone treatment. Taken together, the results suggest that, when encapsulated with chitosan, cells are less susceptible to Se treatment, possibly through a mechanism by which the presence of chitosan attenuates Se-induced activation of ATM and corresponding DNA damage response pathway.