DNA methylation is critical for regulating gene expression in plants and mammals. However, few mutants have been created by demethylation. In this study, AK58 seeds were treated with a DNA methyltransferase inhibitor 5-azacytidine (5-azaC). Mutation variations were generated conferring to heading time, plant height, spike architecture, and yield. Interestingly, we obtained four mutants with a 5-10% increase in thousand grain weight (TGW). Mutant 7128 could flower without vernalization, even 7-10 days earlier than the vernalized wild type AK58. Genetic mapping located the candidate genes to Chr5B and Chr5D. Transcriptome analysis indicated TaVRN1-5B and TaVRN1-5D expressed significantly different between mutant 7128 and wild type AK58. Methylation sequencing further showed that the methylation status of the promoter regions of TaVRN1-5B and TaVRN1-5D were reduced. The increased transcript levels of TaVRN1-5B and TaVRN1-5D promoted the heading of mutant 7128. These results suggested that 5-azaC could activate gene expression through demethylation and produce heritable variations. Our 5-azaC mutant library will be helpful for wheat genetics and breeding.