Spikelet number per panicle (SNP) is one of the most important yield components in rice. RiceENHANCING BIOMASS AND SPIKELET NUMBER(OsEBS), a gene involved in improved SNP and yield, has been cloned from an accession of Dongxiang wild rice. However, the mechanism ofOsEBSincreasing rice SNP is poorly understood. In this study, the RNA-Seq technology was used to analyze the transcriptome of wildtype Guichao 2 andOsEBSover-expression line B102 at the heading stage, and analysis of the evolution ofOsEBSwas also conducted. A total of 5369 differentially expressed genes (DEGs) were identified between Guichao2 and B102, most of which were down-regulated in B102. Analysis of the expression of endogenous hormone-related genes revealed that 63 auxin-related genes were significantly down-regulated in B102. Gene Ontogeny (GO) enrichment analysis showed that the 63 DEGs were mainly enriched in eight GO terms, including auxin-activated signaling pathway, auxin polar transport, auxin transport, basipetal auxin transport, and amino acid transmembrane transport, most of which were directly or indirectly related to polar auxin transport. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analysis further verified that the down-regulated genes related to polar auxin transport had important effects on increased SNP. Analysis of the evolution ofOsEBSfound thatOsEBSwas involved in the differentiation ofindicaandjaponica, and the differentiation ofOsEBSsupported the multi-origin model of rice domestication.Indica(XI) subspecies harbored higher nucleotide diversity thanjaponica(GJ) subspecies in theOsEBSregion, andXIexperienced strong balancing selection during evolution, while selection inGJwas neutral. The degree of genetic differentiation betweenGJandBassubspecies was the smallest, while it was the highest betweenGJandAus. Phylogenetic analysis of the Hsp70 family inO. sativa,Brachypodium distachyon, andArabidopsis thalianaindicated that changes in the sequences ofOsEBSwere accelerated during evolution. Accelerated evolution and domain loss in OsEBS resulted in neofunctionalization. The results obtained from this study provide an important theoretical basis for high-yield rice breeding.