AIMS: In our model, we aimed to explore the cytotoxicity of 5-Aza-2'-deoxycytidine (5-Aza-CdR) against the colorectal cell line, Lovo, and further characterize the possible mechanisms. MAIN METHODS: After Lovo cells were treated with 5-Aza-CdR at different concentrations for different periods of time, the cell viability was examined using an MTT assay and apoptosis was examined using both flow cytometry and DNA laddering. To examine the mechanisms by which Lovo cells respond to 5-Aza-CdR, we measured both caspase 3 activity as well as DNA damage. Western blotting and RT-PCR assays were used to assess the changes in the expression levels of P53, P21(Waf1/Cip1), runt-related transcription factor 3 (RUNX 3), DNA methyltransferases (DNMTs) and matrix metalloproteinases (MMPs). Additionally, we performed gelatin zymography to examine the effects of 5-Aza-CdR on metastasis. KEY FINDINGS: We observed that the growth and survival advantages of Lovo cells were overcome with 5-Aza-CdR treatment at limited concentrations. Mechanistic exploration demonstrated that 5-Aza-CdR was incorporated into the DNA to induce DNA damage in Lovo cells, which was evidenced by activation of P53, P21(Waf1/Cip1) and a caspase-independent cell apoptosis pathway. Also, further experiments preliminarily suggested that 5-Aza-CdR results in the deletion of DNMT 3a and DNMT 3b, but not DNMT 1, which reactivates the expression of RUNX 3. Finally, our data revealed that 5-Aza-CdR potentially reduces the activity and expression of MMP 2. SIGNIFICANCE: These data greatly enhance our understanding of how human cancer cells respond to 5-Aza-CdR and also reveal a new role for 5-Aza-CdR in improving patient outcome in human colorectal cancer.