The granule-bound starch synthase I (GBSSI) encoded by Waxy gene, is responsible for amylose synthesis in endosperm of wheat grains. In the present study, we identified a novel Wx-B1 null mutant line M3-415 from an ethyl methane-sulfonate (EMS) mutagenized population of Chinese tetraploid wheat landrace Jianyangailanmai (LM47). The gene sequence indicates that mutated Wx-B1 is able to encode a complete protein; this is incompatible with the protein profile of SDS-PAGE showing the lack of Wx-B1 protein in the mutant line. Protein structure prediction showed that the amino acid substitution (G470D) was located at the edge of the ADPG binding pocket and might affect the binding of Wx-B1 to the starch granule. We further conducted site-directed mutation to artificially change an amino acid from alanine (A) to threonine (T) at the sequence position of 469 (A469T) ahead mutated site in M3-415. Our results indicated that a single amino acid mutation in Wx-B1 reduces its activity by destroying its starch-binding capacity; this novel mechanism for lacking Wx-1 is firstly reported in wheat. This study also sheds new light on the knowledge of inactivation of the waxy gene and revealed that fine regulation of wheat amylose content by modifying GBSSI activity is possible.