Wheat is a major staple food for more than one-third of the world’s population. Tiller number is an important agronomic trait in wheat, but only few genes have been cloned. Here, we isolated a wheat mutant, tiller number 1 (tn1), with a dramatically reduced tiller number. Through a map-based cloning approach, we cloned the TN1 gene, which encodes an ankyrin repeat protein with transmembrane domain (ANK-TM). A single amino acid substitution (Ala125Thr) in the third conserved ankyrin repeat domain causes the decreased tiller number of tn1 mutants. Resequencing and haplotype analysis showed that TN1 is conserved in wheat landraces and modern cultivars. The expression level of the abscisic acid (ABA) biosynthetic gene TaNCED3 and ABA content are significantly increased in the shoot base and tiller bud of tn1 mutants, which potentially represses tiller bud outgrowth. Taken together, we cloned a wheat tiller number regulatory gene TN1, which is important for wheat tillering.