Hybrid sterility is a major obstacle to the development of superior inter-subspecific hybrids betweenindicaandjaponicasubspecies of Asian-cultivated rice. To overcome hybrid sterility, we introduced fourjaponicaalleles (S-j) atSa,Sc,f5, andS32loci from Nipponbare into 9311 through marker-assisted backcross breeding, and four improved lines were obtained. Three wide-compatibility lines 9311(S5-n), 9311(f5-n), and 9311(S5-n+f5-n) previously constructed were used as control and parental line of hybridization. By crossing with 9311(S5-n+f5-n), two pyramiding lines 9311(Sa+S5-n+f5-n) and 9311(S32+Sa+S5-n+f5-n) were obtained. To evaluate the gene effect of pollen fertility and spikelet fertility, the near-isogenic lines (NILs) harboringS5-n,f5-n,Sa,Sc,f5, orS32and three pyramiding lines withS5-n+f5-n,Sa+S5-n+f5-n, orS32+Sa+S5-n+f5-nwere testcrossed to sixjaponicavarieties. The results showed that single-genef5,f5-n,S32, andS5-ncould significantly improve spikelet fertility in majority ofindica-japonicahybrids. Single-geneSaandSccould significantly improve spikelet fertility in part ofindica-japonicahybrids. Moreover, the pyramiding lines exhibited significantly higher spikelet fertility than the control inindica-japonicahybrids. A significant increase in spikelet fertility (31.4-55%) was observed in three- or four-gene pyramiding lines. Our results provide an effective approach to exploiting heterosis betweenindicaandjaponicasubspecies, which had a profound implication in rice breeding.