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Background The content of stone cells in pears has a great influence on taste. Stone cells are formed by the accumulation of lignin. The treatment of exogenous calcium can affect the lignin synthesis, but this Ca-mediated mechanism is still unclear. In this study, the author performed a comparative transcriptomic analysis of callus of pears (Pyrus x bretschneideri) treated with calcium nitrate Ca (NO3)2 to investigate the role of calcium in lignin synthesis. Results There were 2889 differentially expressed genes (DEGs) detected between the Control and Ca (NO3)2 treatment in total. Among these 2889 DEGs, not only a large number of genes related to Ca single were found, but also many genes were enriched in secondary metabolic pathway, especially in lignin synthesis. Most of them were up-regulated during the development of callus after Ca (NO3)2 treatment. In order to further explore how calcium nitrate treatment affects lignin synthesis, the author screened genes associated with transduction of calcium signal in DEGs, and finally found CAM, CML, CDPK, CBL and CIPK. Then the author identified the PbCML3 in pears and conducted relevant experiments finding the overexpression of PbCML3 would increase the content of pear stone cells, providing potential insights into how Ca treatment enhances the stone cell in pears. Conclusions Our deep analysis reveals the effects of exogenous calcium on calcium signal and lignin biosynthesis pathway. The function of PbCML3 on stone cells formation was verified in pear.