Despite the discovery of numerous oncogenes in colorectal cancer (CRC), the development of associated drugs is limited, posing a significant challenge for CRC treatment. Identification of novel druggable targets is therefore crucial for the therapeutic development of CRC. Here, we report the first investigation on therapeutics targeting the potent oncogene NUCKS1 to suppress cancer progression. NUCKS1-orientated bioinformatics screening of NUCKS1 inhibitors from our library of tRNA fragments originated from medicinal plants identified tRF-T36, a 5' tRNA fragment of tRNAAsn(GUU) of Chinese yew (Taxus chinensis), exhibiting stronger inhibitory effects than taxol against CRC progression. Mechanistically, tRF-T36 binds directly to the 3' UTR of NUCKS1 mRNA to downregulate its expressions via RNAi pathway. High-throughput RNA sequencing indicated that the downregulated NUCKS1 induced by tRF-T36 further inhibits PI3K/Akt pathway, as verified by the significantly efficacy decrease of tRF-T36 mimic in co-treatment with 740Y-P, an agonist of PI3K/Akt pathway. Collectively, our findings emphasize the importance of NUCKS1 as a promising druggable target for CRC. Furthermore, the present study provides the first siRNA sequence, tRF-T36 mimic, as small RNA drug candidate, thereby shedding light on CRC therapeutics.