In pumpkin (Cucurbita moschata), the naked or hull-less seed phenotype has great benefits for breeding this crop for oil or snack use. We previously identified anaked seedmutant in this crop. In this study, we report genetic mapping, identification, and characterization of a candidate gene for this mutation. We showed that the naked seed phenotype is controlled by a single recessive gene (N). The bulked segregant analysis identified a 2.4 Mb region on Chromosome 17 with 15 predicted genes. Multiple lines of evidence suggested thatCmoCh17G004790is the most probable candidate gene for theNlocus which encodes a NAC transcription factor WALL THICKENING PROMOTING FACTOR 1 (CmNST1). No nucleotide polymorphism or structural variation was found in the genomic DNA sequences ofCmNST1between the mutant and the wildtype inbred line (hulled seed). However, the cDNA sequence cloned from developing seed coat samples of thenaked seedmutant was 112 bp shorter than that from the wildtype which is due to seed coat-specific alternative splicing in the second exon of the mutantCmNST1transcript. The expression level ofCmNST1in the developing seed coat was higher in the mutant than in the wildtype during early seed coat development which was reversed later. Transcriptomic profiling with RNA-Seq at different stages of seed development in the mutant and wildtype revealed a critical role ofCmNST1as a master regulator for the lignin biosynthesis pathway during seed coat development while other NAC and MYB transcription factors were also involved in forming a regulatory network for the building of secondary cell walls. This work provides a novel mechanism for the well-characterizedNST1transcription factor gene in regulating secondary cell wall development. The cloned gene also provides a useful tool for marker-assisted breeding of hull-lessC. moschatavarieties.