Genic male-sterile line is an efficient tool for commercial hybrid seed production in pepper; however, so far, only few genes controlling this trait have been cloned. A spontaneous genic male-sterile mutant, msc-1, had been identified and widely used in China, of which the male-sterile trait was proved to be controlled by a single recessive locus. For cloning the gene(s) underlying the msc-1 locus, genome resequencing and comparison analyses were performed between male-sterile and male-fertile lines. According to the genomic variations and genes’ annotations, Capana02g002096 was selected as a candidate gene underlying the msc-1 locus. Capana02g002096 encodes a homolog of AtDYT1, which is a bHLH transcription factor and involves in the early tapetal development. Moreover, a 7-bp deletion was identified in the exon of Capana02g002096, which led to a premature stop codon and may cause a loss-of-function mutation. Further genotyping in the 16C1369AB population containing 1110 plants, a F2 population consisting of 510 plants and 46 inbreed lines revealed that the male-sterile phenotype was co-segregated with the 7-bp deletion. Additionally, real-time PCR analysis revealed that Capana02g002096 was an anther-specific gene and repression of the gene’s expression through VIGS led to male-sterile phenotype. Therefore, based on the evidence at genetic, genomic, transcriptional and posttranscriptional levels, Capana02g002096 was considered as a strong candidate gene underlying the msc-1 locus in pepper and was renamed Msc-1.