In flowering plants, the inflorescence meristem (IM) provides founder cells to form successive floral meristems, the precursors of fruits or seeds. Hence, the activity and developmental progression of IM are critical for yield production in cereal crops. IM size is positively associated with spikelet number in some major cereals, like rice (Oryza sativa) and maize (Zea mays). However, the relationship and regulatory mechanism between IM size and grain yield remain unknown in Triticeae tribe. Here, we report that IM size has a negative correlation with yield traits in barley (Hordeum vulgare), and three FASCIATED EAR (FEA) orthologs, HvFEA2, HvFEA3, and HvFEA4, regulates IM size and spike development and ultimately affect the grain yield. All three HvFEAs are highly expressed in developing spikes, and loss-of-function mutants exhibit enlarged IM size, shortened spike length and decreased spikelet number, which leads to the reduced grain yield. We further reveal that HvFEA4 potentially targets to multiple pathways during reproductive development, including transcriptional control, phytohormones signaling and redox status. Our findings uncover the roles of barley FEA genes in limiting IM size and promoting spikelet formation, which provides insights into yield improvement by manipulating IM activity.