Cancer is the second leading cause of death globally, where nearly 1 in 6 deaths is due to cancer, with 70% of all deaths from cancer occur in low- and middle-income countries. The overall lifetime risk of developing colorectal cancer is 1 in 22 in men and 1 in 24 in women. In this work, we aimed to evaluate the role of temozolomide (TMZ) in controlling colon cancer cells (CRC) via regulating the miRnome. For this purpose, CRC cells (CaCo-2) were treated with 50 µM of TMZ for 48 h. Cell count using trypan test and cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were carried out, and the obtained results indicated a significant decrease in cell count (p = 0.029), and in the cell viability (p = 0.0019). Cell cycle analysis was performed using flow cytometer, and results showed that TMZ arrested CRC cells at G2/M phase. A total of 84 miRNAs were profiled using real time PCR, and the results indicated that TMZ treatment upregulated 15 of 84 miRNAs panel profiled and downregulated the rest. The TMZ-upregulated/downregulated miRNAs were predicted to interact with many epigenetic-related proteins i.e., DNMTs, EZH2, and SUV31H1. This study shed some light on the role of TMZ in regulating the miRnome of CRC and hence in different types of cancers.