Identification of new molecular targets is needed for the treatment of colorectal cancer (CRC). Methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L), an enzyme in the folate cycle, is involved in formate generation and therefore in one-carbon metabolism. Here, we examined the expression and the role of MTHFD1L in CRC progression. Bioinformatics analysis of several public databases showed overexpression of MTHFD1L in CRC tissues as compared to normal tissues. Quantitative real-time PCR and Western blotting revealed that expressions of MTHFD1L RNA and protein were higher in CRC tissues compared to their corresponding normal tissues of CRC patients. Immunohistochemical staining demonstrated higher cytoplasmic MTHFD1L reactivity in tumor tissues compared to paired normal tissues. Further, to determine the functional relevance of MTHFD1L, it was knocked down by an siRNA in CRC cells. Silencing of MTHFD1L inhibited CRC cell proliferation, colony formation, invasion, and migration. Thus, to our knowledge for the first time in the literature, we show that MTHFD1L is involved in CRC progression and that blocking of MTHFD1L decreases the growth of colon cancer cells, thus providing an avenue to target this enzyme with small molecule inhibitors.