Transfer RNA‑derived fragments (tRFs) are a novel class of small non‑coding RNAs that are abundant in various species and have been implicated in human diseases. However, to the best of our knowledge, the role of tRFs in colon cancer has not been explored. In the present study, a comprehensive analysis was conducted to identify the tRFs associated with colon cancer and predict their roles in colon carcinogenesis. tRFs and miRNAs that were differentially expressed (DE) between colon cancer and matched peritumor tissues were identified by small RNA sequencing. DE mRNAs were screened by Gene Expression Profiling Interactive Analysis. DE microRNAs (miRNAs) reported to be associated with colon cancer were selected as key miRNAs. The targets of the DE tRFs and key miRNAs were predicted using MiRanda and RNAhybrid, respectively. The potential targets that were common to and negatively correlated with the DE tRFs and key miRNAs were screened. The DE mRNAs and potential targets were subjected to Gene Ontology and pathway analyses, respectively. In total, 16 DE tRFs, 26 DE miRNAs, and 5,327 DE mRNAs were identified. Five of the 26 DE miRNAs were selected as key miRNAs. DE mRNAs were mainly enriched in cell proliferation‑related processes and pathways. Fifty‑five DE mRNAs were potential targets of both DE tRFs and key miRNAs and were primarily enriched in vitamin metabolic pathways and the cyclic guanine monophosphate‑protein kinase G signaling pathway. Collectively, these results suggest that tRFs may play crucial roles in the development of colon cancer. This study could provide valuable cues for further research into the roles of tRFs in colon cancer.