A lesion-mimic phenotype in rice (Oryza sativa L.) spotted leaf5 (spl5) indicates that wild-type SPL5 negatively regulates cell death and resistance responses.Previously, the spl5 gene was already mapped to the 80-kb region between two markers SSR7 and RM7121 through a map-based cloning approach.Here, we further showed that the spl5 gene was delimitated into a 15.1-kb genomic region by the high-resolution sequence target site (STS) markers.Subsequent sequencing in this region of spl5 mutant revealed that one candidate gene harbored a single-base deletion, resulting in a frame-shift mutation and a premature stop codon.Bioinformatic analysis showed that SPL5 gene encodes a putative splicing factor 3b subunit 3 (SF3b3) and might be involved in splicing reactions of pre-mature RNAs participating in the regulation of cell death and resistance responses.Further analysis showed that wild-type SPL5 did functionally complement the spl5 phenotype.The data presented here clearly indicate that the SPL5 negatively regulates cell death and resistance responses via modulating RNA splicing in plants.