Grain shape and plumpness affect barley yield. Resistant starch has potential application value in food product development. The shrunken endosperm mutant, sex1, was genetically controlled by a single recessive gene. Our results obtained by using genetics, fine mapping, gene isolation, expression validation and transcriptomics suggested that sex1 gene was located in an ~5 kb fragment containing only one ORF (named ORF1) on chromosome arm 6HL which was deleted in sex1 mutant. We thus developed an InDel marker. This marker was co-segregated with the sex1 phenotype in ~8,000 plants. We thus predicated ORF1 was the candidate of sex1 mutant. It encodes a sugar transporter protein and is mainly expressed in kernel. Physiological and biochemical analyses based on a pair of near-isogenic lines (NIL-sex1W and NIL-sex1S) for sex1 gene showed that NIL-sex1S (mutant type) exhibited a full grain phenotype and the water content was significantly higher than that of NIL-sex1W (wild type) during grain development, while the total starch content in mature grains was significantly lower than NIL-sex1W. The total protein, soluble sugar, β-glucan, amylose and resistant starch contents were significantly higher than those of NIL-sex1W. Scanning electron microscope observation showed that the A-type starch granules of NIL-sex1S were significantly reduced. XRD and ATR-FTIR analyses showed that NIL-sex1W and NIL-sex1S had similar infrared spectra and both presented A-type crystallinity. Transcriptomic and metabolomic analyses showed that endosperm shrinkage in barley seemed to be closely related to plant hormone signal transduction, starch, sucrose metabolism, amino acid metabolism and cell apoptosis. Taken together, this study provides new insights into barley grain development and the candidate gene ORF1 should be a key determinant for soluble sugar entering endosperm cells into starch.