Background: Accumulating reports suggest that radiation may change gene expression in cancer cells and promote cell migration and invasion, as well as inducing cancer stem cell (CSC). However, the correlation between these processes and radiation dose has not been shown yet. Therefore, the present study aimed to evaluate the effect of low, medium, and high doses of X-ray on expressing three genes involved in CSC induction in colon cancer cell line (HT-29). Materials and Methods: The cells cultured in flasks were irradiated with X- rays in different doses including 0.1, 2.5, 5, and 10 Gy. Then, the expression of Oct4, CD44, and ALDH1 genes was measured using real-time PCR. PCR efficiency was evaluated for each gene using Linreg PCR software, and relative changes for mRNA were calculated based on the ∆∆Ct method. Results: CD44 gene expression increased equally at all doses. Oct4 and ALDH1 gene expression were not affected by 10 Gy, but low and moderate doses increased them equally. Conclusion: The effects of low and moderate doses on increasing the expression of stem-related genes are equal. In addition, the effect of the high dose on increasing CD44 gene expression was equal to the low and moderate doses.