Natural killer (NK) cells are critical mediators of host immunity to pathogens. Here, we demonstrate that the endoplasmic reticulum stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box-binding protein 1 (XBP1) drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mammalian target of rapamycin signaling pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, and NK cells with c-Myc haploinsufficency phenocopied IRE1α-XBP1 deficiency. c-Myc overexpression largely rescued the proliferation defect in IRE1α-/- NK cells. Like c-Myc, IRE1α-XBP1 also promotes oxidative phosphorylation in NK cells. Overall, our study identifies a IRE1α-XBP1-cMyc axis in NK cell immunity, providing insight into host protection against infection and cancer.