Increasing grain yield is an endless challenge for cereal crop breeding. In barley, grain number is mainly controlled by Six-rowed spike 1 (Vrs1) that encodes a homeodomain leucine zipper class I transcription factor. However, little is known about the genetic basis of grain size. Here we show that extreme suppression of lateral florets contributes to enlarged grains in deficiens barley. Through a combination of fine mapping and resequencing deficiens mutants we have identified that a single amino acid substitution at a putative phosphorylation site in VRS1 is responsible for the deficiens phenotype. deficiens mutant alleles confer an increase in grain size, reduction in plant height and a significant increase in thousand grain weight in contemporary cultivated germplasm. Haplotype analysis revealed that barley carrying the deficiens allele (Vrs1.t1) originated from two-rowed types carrying the Vrs1.b2 allele, predominantly found in germplasm from Northern Africa. In situ hybridization of histone H4, a marker for cell cycle or proliferation, showed weaker expression in the lateral spikelets compared to central spikelets in deficiens. Transcriptome analysis revealed that a number of histone superfamily genes were upregulated in the deficiens mutant suggesting that enhanced cell proliferation in the central spikelet may contribute to larger grains. Our data suggest that grain yield can be improved by suppressing the development of specific organs that are not positively involved in sink/source relationships.