A major QTL for heading date, qHD5, was fine-mapped to a 52.59-kb region on the short arm of rice chromosome 5. Heading date (HD) is one of the most important traits that enables rice to adapt to seasonal differences and specific growth conditions in diverse growing regions. In this study, a major-effect quantitative trait locus (QTL), qHD5, was resolved as a single Medelian factor that causes NIL(BG1) and NIL(XLJ) (two near-isogenic lines (NILs) used in our study) to have at a minimum of 10-day difference in HD under both long-day and short-day conditions in rice. qHD5 was initially mapped to a 309.52-kb genomic region in our previous study. Here, using an advanced BC4F3 population and map-based cloning, we further narrowed the location of qHD5 to a 52.59-kb region between the H71 and RD502 markers. Sequence analysis revealed that Os05g03040, which putatively encodes an AP2 (APETALA2) transcription factor, has six single nucleotide polymorphisms (SNPs) between NIL(BG1) and NIL(XLJ). On this basis, this gene was concluded to be the most probable candidate gene for qHD5. Our results also showed that Hd3a, RFT1, Hd1, Ehd1, and Ghd7 were differentially expressed in the two NILs. Moreover, qHD5 was found to affect yield-related traits such as flag leaf width, flag leaf length, branch number, and 1000-grain weight.