ABSTRACT: Polygalacturonase (PG) is an essential hydrolytic enzyme responsible for pectin degradation and thus plays an important role in fruit softening and other cell separation processes. PG protein is encoded by a multigene family, however, the members of PG gene family in kiwifruit (Actinidia chinensis) have not been extensively identified. In this study, a total of 51 AcPG genes in kiwifruit genome were identified. They are phylogenetically clustered into seven clades, and of them AcPG4 and AcPG18 with other known PG genes involved in fruit softening from peach, pear, papaya and melon form a small cluster together. The members of kiwifruit PG gene family consist of three to nine exons and two to eight introns, and their exon/intron structures are generally conserved in all clades except the clade D and E. During fruit softening of kiwifruit 'Donghong' under ambient temperature, cell wall modifying enzymes, including PG, PL (pectate and pectin lyases), and PE (pectinesterase, also known as pectin methylesterase, PME) showed a different activity profile, and of them, PG and PE activities largely correlated with the change of pectin content and firmness. Moreover, only 11 AcPG genes were highly or moderately expressed in softening fruit, and of which three AcPG genes (AcPG4, AcPG18, and AcPG8, especially the former two) has been found to strongly correlate with the profile of PG activity and pectin content, as well as fruit firmness, suggesting that they maybe play an important role in fruit softening. Thus, our findings not only benefit the functional characterization of kiwifruit PG genes, but also provide a subset of potential PG candidate genes for further genetic manipulation.