Hybrid rice has made considerable contributions to achieve the ambitious goal of food security for the world population. Hybrid rice fromindica/xianandjaponica/geng subspecies shows much higher heterosis and is thereby an important innovation in promoting rice production in the next decade. However, such inter-subspecific hybrid rice has long suffered from serious hybrid sterility, which is a major challenge that needs to be addressed. Here we performed a genome design strategy to produce fertile inter-subspecific hybrid by creation of wide-compatibility varieties that are able to overcome hybrid sterility. Based on combined genetic analyses in twoindica-japonicacrosses, we determined that four hybrid sterility loci,S5,f5,pf12 and Sc, were the major QTLs for inter-subspecific hybrid sterility and were the minimal targets that need to be manipulated in breeding process. We therefore cloned the pf12 locus, one of the most effective loci for hybrid male sterility, by map-based cloning. Artificial disruption of pf12A gene at this locus successfully rescued hybrid fertility. We further dissected the genetic basis of wide compatibility using three pairwise crosses from a wide-compatibility variety Dular and representative indica and japonica varieties. On this basis, we constructed and assembled different combinations of naturally compatible alleles of four loci,S5,Sc,pf12, and f5, and the improved lines can fully recover pollen and embryo-sac fertility in testcrossed F1s, thereby completely fulfilling the demands of inter-subspecific hybrid spikelet fertility in production. This breeding scheme would facilitate redesign of future inter-subspecific hybrid rice with higher yield potential.