Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide, and its occurrence can be ascribed to genetic susceptibility. Mitochondrial DNA 4977-bp (mtDNA 4977), as the most described mtDNA deletion, has been long proposed to be involved in various types of cancers. However, a few studies on mtDNA 4977-bp deletion in Iranian patients with CRC have been reported. The current study aimed to determine mtDNA 4977 frequency in CRC and its association with cancer susceptibility. We conducted a case-control study in which a total of 26 patients with CRC, 26 tumor tissues, adjacent normal tissues, peripheral blood samples, and peripheral blood samples from 50 healthy subjects were included. mtDNA 4977 was detected using multiplex PCR technique and direct DNA sequencing. Real-time PCR was also used to determine deletion levels. mtDNA 4977 was observed in six patients (23.07%), four (15.3%) in both tumor and matched surrounding normal tissues, and two (7.69%) in adjacent normal tissues, but not detected in both patients and control samples in peripheral samples. A significant difference was found between mtDNA 4977 deletion in tumoral and adjacent normal tissues (P=0.001). No relation was observed between mtDNA 4977 and categorical variables, including age and gender, and tumor stage. The analysis confirmed no association between the mtDNA 4977-bp deletion and susceptibility to colorectal cancer in Iranian patients. However, more extensive studies are required to confirm or reject these findings.