The polymerization or depolymerization of the cytoskeleton is very important in various aspects of plant development. ADF (actin-depolymerizing factor) is a typical actin binding protein that can mediate the polymerization or depolymerization of the actin cytoskeleton, but its role in fruit ripening remains unclear. Here, we characterized an ADF gene, PuADF. The expression of PuADF was evaluated in young leaves, stems, flowers, and roots as well as in fruits. The expression of PuADF was induced by ethephon treatment but inhibited by 1-MCP treatment. To explore the network of PuADF function in ‘Nanguo’ pear fruits ripening, we screened a cDNA library from ‘Nanguo’ pear fruits using PuADF as bait. Two potential interactors of PuADF were identified, PuAS (Asparagine Synthetase) and PuDAD1 (Defender Against Death 1), both of which have been associated with PCD (Programmed Cell Death). This direct interaction was further confirmed by yeast two-hybrid analyses. The expression levels of PuAS and PuDAD1 were affected by ethylene. Our results indicated that PuADF is involved in ethylene-mediated fruit ripening and might be related to PCD during fruit ripening. The possible mechanisms are discussed.