We recently reported that the tumor suppressor Runt-related transcription factor 3 (RUNX3) is silenced in colorectal cancer cells via oxidative stress-induced hypermethylation of its promoter. The resulting downregulation of RUNX3 expression influences cell proliferation. Activation of the Akt signaling pathway is also associated with cell survival and proliferation; however, the effects of oxidative stress on the relationship between RUNX3 and Akt signaling are largely unknown. Therefore, this study investigated the mechanisms involved in cell proliferation caused by oxidative stress-induced silencing of RUNX3. The levels of RUNX3 mRNA and protein were downregulated in response to treatment of the human colorectal cancer cell line SNU-407 with H2O2. Treatment of the cells with H2O2 also upregulated Akt mRNA and protein expression, and inhibited the binding of RUNX3 to the Akt promoter. The inverse correlation between the expression levels of RUNX3 and Akt in H2O2-treated cells was also associated with nuclear translocation of β-catenin and upregulation of cyclin D1 expression, which induced cell proliferation. H2O2 treatment also increased the binding of β-catenin to the cyclin D1 promoter. The results presented here demonstrate that reactive oxygen species silence the tumor suppressor RUNX3, enhance the Akt-mediated signaling pathway, and promote the proliferation of colorectal cancer cells.