Sorghum [Sorghum bicolor (L.) Moench] is an important crop for food, feed, and fuel production. Particularly, sorghum is targeted for cellulosic ethanol production. Extraction of cellulose from cell walls is a key process in cellulosic ethanol production, and understanding the components involved in cellulose synthesis is important for both fundamental and applied research. Despite the significance in the biofuel industry, the genes involved in sorghum cell wall biosynthesis, modification, and degradation have not been characterized. In this study, we have identified and characterized three allelic thick leaf mutants (thl1, thl2, and thl3). Bulked Segregant Analysis sequencing (BSAseq) showed that the causal mutation for the thl phenotype is in endo-1,4-β-glucanase gene (SbKOR1). Consistent with the causal gene function, the thl mutants showed decreased crystalline cellulose content in the stem tissues. The SbKOR1 function was characterized using Arabidopsis endo-1,4-β-glucanase gene mutant (rsw2-1). Complementation of Arabidopsis with SbKOR1 (native Arabidopsis promoter and overexpression by 35S promoter) restored the radial swelling phenotype of rsw2-1 mutant, proving that SbKOR1 functions as endo-1,4-β-glucanase. Overall, the present study has identified and characterized sorghum endo-1,4-β-glucanase gene function, laying the foundation for future research on cell wall biosynthesis and engineering of sorghum for biofuel production.