•Intracellular Ca2+ responses to pear scab infection in two cultivars were analyzed.•Mechanisms underpinning scab-induced intracellular Ca2+ changes were elaborated.•Six Ca2+ sensor genes participate in pear scab resistance in the resistant cultivar. In this study, we investigated the role of Ca2+ signaling in pear (Pyrus. bretschneideri Rehd) in response to pear scab (Venturia nashicola Tanak et Yamamota) infection. We analyzed the responses of intracellular Ca2+ and expression changes of Ca2+ sensor-related genes in two pear cultivars of differing resistance (the resistant ‘Huangguan’ and the susceptible ‘Xuehua’) following V. nashicola infection by using fluorescence microscopy, microplate spectrophotometry, and real-time quantitative PCR assays. Over a 60 min treatment period, V. nashicola induced elevated Ca2+ fluorescence in cell protoplasts (loaded with Fluo-3 AM) from fruit callus cultures of both pear cultivars, but the Ca2+ change patterns differed. Both LaCl3 (a plasma membrane Ca2+ channel inhibitor) and EGTA (an extracellular Ca2+ chelator) inhibited Ca2+cyt in both cultivars, while BAPTA-AM (an intracellular Ca2+ chelator) showed no inhibitory effect. The relative expression levels of PbCaM1, PbCaM5, PbCBL1, PbCIPK2, PbCPK1, and PbCPK2 in ‘Huangguan’ pear were significantly upregulated following infection and peaked at 72 post-infection hour. PbCPK1 and PbCPK2 underwent the most intense expression changes of these six genes tested. In ‘Xuehua’ pear, only PbCBL1 and PbCIPK2 were significantly upregulated following infection, while the remaining genes were downregulated to varying degrees. In conclusion, V. nashicola infection induced Ca2+ signaling in pear cells, and changes in Ca2+cyt were mainly due to extracellular Ca2+ influx. In ‘Huangguan’ pear, all tested genes related to Ca2+ sensors responded to V. nashicola infection, and might participate in resistance to the scab pathogen.