Pungency in peppers is due to the presence of the alkaloid capsaicin and its analogues, collectively known as capsaicinoids. These compounds are only produced in the Capsicum genus and function as deterrents to mammals from consuming the pepper fruits. Pungency in pepper is qualitatively controlled by the Pun1 locus, which encodes a putative acyltransferase enzyme. Mutations in the Pun1 gene result in a loss of pungency, and several Pun1 loss-of-function alleles have been identified in sweet peppers to date (pun11–3). However, variations in pun1 alleles have not been completely elucidated. In the present study, we report a new type of loss-of-function pun1 allele, named pun14, in a Japanese sweet pepper cultivar, ‘Nara Murasaki’ (C. annuum). Sequence analysis at the Pun1 locus revealed that this type of Pun1 allele is caused by a single adenine nucleotide insertion in the second exon region. This insertion is unique to ‘Nara Murasaki’ and is not present in wild-type Pun1. This insertion causes a frameshift mutation and a change in the amino acid sequence, resulting in a truncated protein. The results of gene expression analysis indicated that the expression of Pun1 in ‘Nara Murasaki’ was hardly detectable, while the transcripts of this gene were strongly expressed in a pungent cultivar. In a co-segregation test, the pun14 genotype perfectly co-segregated with non-pungency in 103 F2 population plants of a cross between ‘Nara Murasaki’ and a pungent cultivar. ‘Nara Murasaki’ and a DNA marker to distinguish the pun14 allele will be informative for understanding the domestication process of sweet peppers.