PURPOSE: Colorectal cancer is one of the most common and deadliest cancer types worldwide. In the last years, changes in the mitochondrial DNA (mtDNA) copy number have been described to correlate with the prognostic outcome for colorectal cancer patients by impacting different tumorigenic properties. One key regulator of mtDNA is the mitochondrial transcription factor A (TFAM) that acts as a limiting factor of mtDNA copy number. Here, we investigated the effect of TFAM deficiency on mtDNA and tumorigenic properties in the human colorectal cancer cell line SW480. METHODS: TFAM expression was stably downregulated in the colorectal cancer cell line SW480 using the CRISPR-Cas9 approach. To dissect the molecular alterations induced by deletion of TFAM, RNA sequencing and gene set enrichment analysis was performed on TFAM-wild-type and TFAM-deficient SW480 cells. Functional consequences of TFAM downregulation were assessed in cellular assays. RESULTS: We showed that TFAM deficiency leads to decreased mtDNA copy number and reduced expression of mtDNA-encoded genes. TFAM-deficient cells also revealed higher activity of senescence-associated β-galactosidase and decreased cell growth parameters. Moreover, RNA sequencing showed that the expression of cyclin dependent kinase inhibitor 1A (CDKN1A/p21) is significantly increased in TFAM-deficient cells. CONCLUSION: Our results suggest that TFAM-induced changes of the mitochondrial genome lead to upregulated CDKN1A/p21 expression in colorectal cancer cells identifying p21 as a new possible linker between mitochondria and nucleus.