As a signaling molecule in plants and animals, calcium plays important roles in cell growth and development. A concentration gradient of calcium in the apex of tip-growing cells regulates polarized growth. In this study, we cloned the gene encoding a calcineurin B subunit protein, MdCBL5, in “Ralls Janet” apple. We found that MdCBL5 was strongly expressed in pollen and that the encoded protein contained the conserved calcineurin EF hand domain. Antisense-mediated silencing of MdCBL5 disrupted the calcium ion concentration gradient in the pollen tube apex and inhibited pollen tube growth. During the self-incompatibility response in apple, S-RNase uptake disrupts the calcium gradient in the pollen tube apex and inhibits pollen tube growth. Quantitative PCR analysis indicated that MdCBL5 was downregulated in pollen tubes treated with S-RNase. We confirmed the interaction between MdCBL5 and S-RNase using yeast two-hybrid, pull-down, and bimolecular fluorescence complementation (BiFC) experiments. These results show that MdCBL5 is a calcium signaling factor that interacts with S-RNases and regulates pollen tube growth and is itself likely regulated by S-RNase during the self-incompatibility response.