Mitochondrial ribosome proteins (MRPs), which are encoded by the nuclear genomic DNA, are important for mitochondrial-encoded protein synthesis and mitochondrial function. Emerging evidence suggests that several MRPs also exhibit important extra-mitochondrial functions, such as involvement in apoptosis, protein biosynthesis, and signal transduction. In this study, we demonstrate a significant role of MRP L35 (MRPL35) in colorectal cancer (CRC). The expression of MRPL35 was higher in CRC tissues than in matched cancer-adjacent tissues and higher in CRC cells than in normal mucosal epithelial cells. Higher MRPL35 expression in CRC tissue correlated with shorter overall survival for CRC patients. In vitro, down-regulation of MRPL35 led to increased production of reactive oxygen species (ROS) together with DNA damage, loss of cell proliferation, G 2 /M arrest, a decrease in mitochondrial membrane potential, apoptosis, and autophagy induction. MRPL35 knockdown inhibited tumor proliferation in a CRC xenograft nude mouse model. Furthermore, overexpression of MRPL35 or treatment of cells with the ROS scavenger, N-acetyl cysteine, abrogated ROS production, cell cycle arrest, and apoptosis in vitro. These findings suggest that MRPL35 plays an essential role in the development of CRC and may be a potential therapeutic target for CRC. (Am J Pathol 2019, 189: 1105e1120; https:// Colorectal cancer (CRC), characterized by fast progression and poor prognosis, is one of the most common malignancies in the world. 1 The mechanisms of CRC development are still poorly understood. Although surgery is the most effective therapy for CRC, chemotherapy and radiotherapy are still indispensable. 2 Side effects and drug resistance are common issues with most of these treatments reducing their efficacy. As a consequence, CRC is in urgent need of markers for early diagnosis and of intervention targets for effective therapy.Mitochondria have a central role in cellular energy metabolism by oxidative phosphorylation, regulation of apoptosis, autophagy, and cell death. 3e5 Defective mitochondrial functions have been thought to be possible underlying mechanisms for cancer. 6,7 Mitochondria have their own DNA and ribosomes (mitoribosomes), which produce 13 proteins essential for oxidative phosphorylation. 8 Human mitoribosomes sediment as 55S particles, consisted of a 28S small subunit, formed by a 12S rRNA and 29 mitochondrial ribosome proteins (MRPs), and a 39S large subunit, formed by a 16S rRNA and 50 MRPs. 9,10 The approximately 70 MRPs are encoded by nuclear genomic DNA, synthesized in the cytosol,