Hybrid sterility is a major form of postzygotic reproductive isolation that restricts gene flow between populations. Cultivated rice (Oryza sativa L.) consists of two subspecies, indica and japonica; inter-subspecific hybrids are usually sterile. We show that a killer-protector system at the S5 locus encoded by three tightly linked genes [Open Reading Frame 3 (ORF3) to ORF5] regulates fertility in indica-japonica hybrids. During female sporogenesis, the action of ORF5+ (killer) and ORF4+ (partner) causes endoplasmic reticulum (ER) stress. ORF3+ (protector) prevents ER stress and produces normal gametes, but ORF3- cannot prevent ER stress, resulting in premature programmed cell death and leads to embryo-sac abortion. Preferential transmission of ORF3+ gametes results in segregation distortion in the progeny. These results add to our understanding of differences between indica and japonica rice and may aid in rice genetic improvement.