Transfer RNA-derived fragments have specific biological roles. However, it is still not characterized what factors are responsible for generation of 5′-tRHs in certain conditions, such as metabolic disease and maturation of reproductive cells. Here, we report that Inositol-requiring enzyme 1α (IRE1α), a major ER stress sensor protein, cleaves specifically anticodon stem-loop region of tRNAGly(GCC) and produces 5′-tRHs. Using an RNA-seq-based approach, we identified cleavage sites in tRNAGly(GCC), generating 5′-tRHs in KGN cells (human ovarian granulosa cells) in an IRE1α-expression dependent manner. In vitro cleavage analyses further supported that IRE1α generates 5′-tRHs from tRNAGly(GCC) (5′-tRH-GlyGCC) with highly selective target discrimination. The production of 5′-tRH-GlyGCC was promoted upon endoplasmic reticulum (ER) stress, which induced IRE1α expression, in KGN cells as well as other cancer cell lines such as HeLa and HepG. In addition, transfection of synthetic 5′-tRH-GlyGCC mimics promoted survival of KGN and HeLa cells; this effect required expression of HNRNPM and HNRNPH2, which were identified as binding proteins of 5′-tRH-GlyGCC.