Objective. The purpose is to study the effect of tRNA-derived fragments (tRFs) on pan-cancer through bioinformatics. Methods. The expression information of tRF-20-S998LO9D, a type of tRF-5, was retrieved through MINTbase in pan-cancer and verified by qPCR. We preliminarily explored the effect of tRF-20-S998LO9D on cell proliferation in breast cancer and lung cancer cell lines. Then an online KM-plotter provided by OncotRF was used to discover the prognostic significance. GO/KEGG analyses were executed to predict the potential mechanism of tRF-20-S998LO9D in cancer. Results. We found that tRF-20-S998LO9D was highly expressed in a variety of cancers like breast invasive carcinoma, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, lung squamous cell carcinoma, pheochromocytoma and paraganglioma, and uterine corpus endometrial carcinoma. Inhibition of tRF-20-S998LO9D led to reduced cell proliferation in breast cancer (MCF-7) and lung squamous cell carcinoma (SK-MES-1) cells. Elevated tRF-20-S998LO9D indicated poor prognosis in a variety of cancers. tRF-20-S998LO9D might be involved in multiple cancer-related pathways. Conclusion. We concluded that tRF-20-S998LO9D was upregulated and negatively correlated with prognosis of a variety of cancers. It may be a potential cancer-promoting marker in pan-cancer.