SummaryGlobal warming is a major abiotic stress factor, which limit rice production. Exploiting the genetic basis of the natural variation in heat resistance at different reproductive stages among diverse exotic Oryza germplasms can help breeding heat‐resistant rice cultivars. Here, we identified a stable quantitative trait locus (QTL) for heat tolerance at the heading stage on chromosome 5 (qHTH5) in O. rufipogon Griff. The corresponding gene, HTH5, pertains to the pyridoxal phosphate‐binding protein PLPBP (formerly called PROSC) family, which is predicted to encode pyridoxal phosphate homeostasis protein (PLPHP) localized to the mitochondrion. Overexpression of HTH5 increased the seed‐setting rate of rice plants under heat stress at the heading stage, whereas suppression of HTH5 resulted in greater susceptibility to heat stress. Further investigation indicated that HTH5 reduces reactive oxygen species accumulation at high temperatures by increasing the heat‐induced pyridoxal 5'‐phosphate (PLP) content. Moreover, we found that two SNPs located in the HTH5 promoter region are involved with its expression level and associated with heat tolerance diversity. These findings suggest that the novel gene HTH5 might have great potential value for heightening rice tolerance to heat stress to the on‐going threat of global warming.