Lignin deposition in stone cells is a critical factor that limits pear fruit quality, affecting their market value. Calcium ions (Ca ) play an essential role in lignin biosynthesis during fruit stone cell production. However, the genetic mechanisms underlying the Ca regulated lignin synthesis in stone cell formation are not fully understood. In this study, we identified an NAC transcription factor (TF) PuNAC21, which is repressed by CaCl treatment. PuNAC21 bound directly to the lignin biosynthesis gene peroxidase 42-like (-like) promoter, Ca reduced pear fruit stone cell production dependent on PuNAC21 positively regulating -like expression. Furthermore, PuNAC21 directly regulated the expression of , a TF involved in lignin biosynthesis by binding to -like and caffeoyl-CoA-O-methyltransferase 1() promoters. Moreover, PuNAC21 interacted with PuDof2.5 to form a transcriptional regulatory module, lowering the transcription of -like and after Ca treatment, which contributed to decrease pear stone cells production. Our results revealed Ca-induced PuNAC21-PuDof2.5-PuPRX42-like/PuCCoAOMT1 regulatory module inhibited lignin biosynthesis, giving important insights into reducing the stone cell content in pears via molecular breeding.