Background <p>Adenosine-to-Inosine (A-to-I) modification is one of the most common transfer RNA (tRNA) modifications in humans. However, the role of A-to-I tRNA modification in colorectal cancer (CRC) remains poorly understood.</p> Methods <p>tRNA modification was profiled by LC-MS in paired CRC and adjacent normal tissues (<i>N</i> = 70). The clinical significance of adenosine deaminase tRNA specific 2 (ADAT2) was evaluated using in-house (<i>N</i> = 157) and TCGA cohorts (<i>N</i> = 283). The function of ADAT2 in CRC was determined in intestine-specific ADAT2 knockout mice. Mechanism of ADAT2 was assessed by integrated RNA-sequencing, tRNA-sequencing, and ribosome-sequencing analyses.</p> Results <p>Among 32 tRNA nucleotide modifications, A-to-I modification is the top enriched tRNA modification in CRC tumors compared to paired adjacent normal tissues (<i>P</i> &lt; 0.001). Consistently, A-to-I modification enzyme ADAT2 is elevated in CRC and associated with poor patient survival in independent patient cohorts. Functionally, ADAT2 overexpression promotes malignant phenotypes in CRC cells and patient-derived CRC organoids, whereas ADAT2 knockout exerts opposite effects. Intestine-specific ADAT2 knockout mice showed attenuated colorectal tumorigenesis. Integrated sequencing identified that ADAT2 boosts translation efficiency of genes highly dependent on A-to-I codons, specifically enriched in WNT/β-catenin signaling. We revealed HDAC7 as a downstream target, whereby ADAT2 promotes HDAC7 translation in an A-to-I dependent fashion. HDAC7 interacts with β-catenin, leading to its activation and nuclear translocation. For translational value, ADAT2 promotes chemoresistance in CRC, and targeting ADAT2 by VNP-encapsulated ADAT2-siRNA promoted Oxaliplatin and 5-Fluorouracil efficacy to suppress CRC growth.</p> Conclusions <p>ADAT2-driven tRNA A-to-I modification promotes CRC tumorigenesis and chemoresistance via HDAC7-WNT/β-catenin axis, and is an independent prognostic factor.</p>