The barley (Hordeum vulgare L.) spike consists of one central and two lateral spikelets at each rachis node. In two-rowed barley, only the central spikelet is fertile, the lateral spikelets also produce grain while in six-rowed barley. Five SIX-ROWED SPIKE genes (VRS1-5) have been identified as regulators of lateral spikelet fertility in barley, but the underlying genetic mechanisms of these VRS genes remain unclear. In this study, we conducted a detailed observation of the development process of the lateral spikelets in two-rowed barley and performed comparative transcriptome analysis to investigate gene expression differences between vrs1-5 mutants and wild-type spikelets. This revealed the differences in the downstream pathways regulated by the VRS genes in lateral spikelet development and the correlation of the effects of different VRS genes on lateral spikelet fertility. Using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq), we identified 213 direct downstream genes of VRS1, including those involved in energy metabolism, hormone pathways, and transcription factors. We also discovered that VRS1 directly binds to the D-class gene HvMADS13 to regulate spikelet fertility. Further analysis of the six-rowed barley accessions revealed that a 1 bp deletion in the C-terminus of VRS1 disrupts its ability to repress transcription, leading to fertility in the lateral spikelets.