Background and Objectives:Colon cancer (CC) is the second most common cancer in Saudi Arabia, and the number of new cases is expected to increase by 40% by 2040. Sixty percent of patients with CC are diagnosed in the late stage, causing a reduced survival rate. Thus, identifying a new biomarker could contribute to diagnosing CC in the early stages, leading to delivering better therapy and increasing the survival rate.Materials and Methods: HSPB6expression was investigated in extracted RNA taken from 10 patients with CC and their adjacent normal tissues, as well as in DMH-induced CC and a colon treated with saline taken from a male Wistar rat. Additionally, the DNA of the LoVo and Caco-2 cell lines was collected, and bisulfite was converted to measure the DNA methylation level. This was followed by applying 5-aza-2′-deoxycytidine (AZA) to the LoVo and Caco-2 cell lines for 72 h to see the effect of DNA methylation onHSPB6expression. Finally, the GeneMANIA database was used to find the interacted genes at transcriptional and translational levels withHSPB6.Results:We found that the expression ofHSPB6was downregulated in 10 CC tissues compared to their adjacent normal colon tissues, as well as in the in vivo study, where its expression was lower in the colon treated with the DMH agent compared to the colon treated with saline. This suggests the possible role ofHSPB6in tumor progression. Moreover,HSPB6was methylated in two CC cell lines (LoVo and Caco-2), and demethylation with AZA elevated its expression, implying a mechanistic association between DNA methylation andHSPB6expression.Conclusions: Our findings indicate thatHSPB6is adversely expressed with tumor progression, and its expression may be controlled by DNA methylation. Thus,HSPB6could be a good biomarker employed in the CC diagnostic process.