A waxy (Wx) null mutant was identified from an ethyl methane sulfonate (EMS)-mutagenized barley population. The difference in the Wx genes between the mutant (M2-1105) and the wild-type Golden Promise (GP) plant were two single-base mutations. The first mutation was located at 1,086 bp (G1086A) at the 3’ splicing site of the fourth intron. This destroyed the splicing receptor site by converting GU to AU, which resulted in abnormal RNA splicing and the inactivation of the M2-1105 Wx gene. The second site, located at 2,424 bp (A2424G), was a synonymous mutation in the ninth intron and did not affect the Wx gene. We evaluated 62 cDNA transcripts of M2-1105 that were summarized as two types. The type I transcript had a 6 bp deletion on the 5’ of exon 5, which resulted in two missing amino acids compared to GP. In the type II transcript, the fourth intron was incorrectly cleaved and retained, which resulted in the premature termination of the barley Wx gene. The loss of Wx-1 decreased the amylose content and changed the starch granule morphology and thermodynamic properties of the barley mutant line. This work sheds lights on the Wx-1 gene inaction mechanism and provides a valuable germplasm to improve barley starch.