Abstract: Against the background of global population growth and increasing pressure on arable land resources, enhancing crop yield per unit area has become a critical objective in agricultural research. As one of the world’s staple food crops, rice yield directly impacts food security. Tiller number is one of the key factors determining the yield of rice, as it directly influences the number of effective panicles. In this study, using ethyl methanesulfonate (EMS) mutagenesis in the background of the japonica rice cultivar Shennong 9816 , induced a rice mutant with reduced tiller number, designated less tiller number 4-1 (ltn4-1), was obtained. Through map-based cloning, the target gene was localized to a 106 kb region between markers G5-10A2a and SN5-1 on chromosome 5. In addition to decreased tiller number, mutation in LTN4 affected leaf width. The ltn4-1 mutant exhibited significantly widened leaves and bulging leaf veins. The number of large vascular bundles and small vascular bundles between large ones in the flag leaf, second uppermost leaf, and third uppermost leaf, were markedly increased in the mutant. Additionally, both the cell number of cells surrounding the vascular bundles were significantly increased in ltn4-1 , and the cell area was enlarged. Agronomic trait analysis revealed that the single panicle weight and tiller number of ltn4-1 were significantly lower than those of the wild type. Furthermore, transcriptome high-throughput sequencing and real-time quantitative RT-PCR results indicated that among the differentially expressed genes between the wild type and the mutant, the expression of 14 known genes associated with leaf width and tiller number regulation, including THIS1, were altered.