Transfer RNA halves (tRHs) have various biological functions. However, the biogenesis of specific 5'-tRHs under certain conditions remains unknown. Here, we report that inositol-requiring enzyme 1α (IRE1α) cleaves the anticodon stem-loop region of tRNAGly(GCC) to produce 5'-tRHs (5'-tRH-GlyGCC) with highly selective target discrimination upon endoplasmic reticulum (ER) stress. Levels of 5'-tRH-GlyGCC positively affect cancer cell proliferation and modulate mRNA isoform biogenesis both in vitro and in vivo; these effects require co-expression of two nuclear ribonucleoproteins, HNRNPM and HNRNPH2, which we identify as binding proteins of 5'-tRH-GlyGCC. In addition, under ER stress in vivo, we observe simultaneous induction of IRE1α and 5'-tRH-GlyGCC expression in mouse organs and a distantly related organism, Cryptococcus neoformans. Thus, collectively, our findings indicate an evolutionarily conserved function for IRE1α-generated 5'-tRH-GlyGCC in cellular adaptation upon ER stress.