In order to identify host factors which interact with the movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV), ACLSV MP was cloned into the bait vector pGBKT7 and used to screen a cDNA library of Malus sylvestris cv. R12740-7A, which had previously been constructed by yeast two-hybrid sequencing transformation. The protein functions of the identified host factors were determined according to their gene annotations in GenBank. The result showed that the bait plasmid pGBKT7-MP showed no virulence or self-activating effect on yeast strain Y2H Gold. Sixty-nine interactor proteins were identified, which were divided into the following 10 classes according to their described functions: hydrolases; pathogenesis-related proteins; DNA binding proteins; phosphatases; ligases; proteins with catalytic activity; phenylalanine ammonialyases; peroxidases; NAD binding proteins; and proteins of unknown function. Bioinformatic analysis of gene homology suggested that phosphatases, pathogenesis-related proteins and glyceraldehyde-3-phosphate dehydrogenase A may play an important role in the interaction between virus and host. This study may provide a theoretical basis for the further study of viral pathogenesis and virus-host interaction mechanisms.