The hulless barley planted in Tibetan area, China (also called highland barley) is well known for their high β-glucan content. This study investigated the proteomics of three highland barley cultivars differing in grain β-glucan content (high, BH; middle, BM and low, BL) to identify the proteins involved in β-glucan synthesis and accumulation and understand the mechanisms underlying the difference of β-glucan content among these cultivars. Totally 38, 167 and 177 differentially expressed proteins (DEPs) were detected from BH VS BM, BH VS BL and BM VS BL comparisons, respectively. These DEPs are mainly enriched in the categories related to β-glucan biosynthesis, carbohydrate metabolism, glucose and sucrose dissimilation. Many glycosidases and glycosyltransferases proteins involved in polysaccharide catabolism were significantly down-regulated in BH and BM cultivars in comparison with BL. The expression of some glycosidases proteins was further confirmed by Parallel Reaction Monitoring (PRM)-MS analysis. These results provide new insights into the proteins that might influence β-glucan levels in highland barley cultivars, which could be helpful for genetic improvement of β-glucan content in barley.