Self-incompatibility (SI) is a reproductive isolation mechanism in flowering plants. Plants in the Solanaceae, Rosaceae and Plantaginaceae belong to the gametophytic self-incompatibility type. S-RNase, which is encoded by a female-specific gene located at the S locus, degrades RNA in the pollen tube and causes SI. Recent studies have provided evidence that S-RNase is transported non-selectively into the pollen tube, but have not specified how this transportation is accomplished. We show here that the apple (Malus domestica) MdABCF protein, which belongs to group F of the ABC transporter family, assists in transportation of S-RNase into the pollen tube. MdABCF is located in the pollen tube membrane and interacts with S-RNase. S-RNase was unable to enter the pollen tube when MdABCF was silenced by antisense oligonucleotide transfection. Our results show that MdABCF assists in transportation of either self or non-self S-RNase into the pollen tube. Moreover, MdABCF coordinates with the cytoskeleton to transport S-RNase. Blockage of S-RNase transport disrupts self-incompatibility in this system.