Strawberry fruits are perishable due to fruit softening occurring during storage and marketing, but the mechanism by which this occurs is not clear. In this study, the possible mechanism of softening is explored by comparative studies on the changes of fruit firmness, pectate lyase (PL) activities, and relative expression levels of PL genes between two strawberry cultivars. The results indicate that a method for the determination of PL activity in the strawberry fruit was established. The activity of PL was at a low level in the small green fruit to white fruit stage, and maintained at a high level in the ripening and softening stage. The PL gene was specifically expressed in fruit and mainly in the ripening stage. PL expressions were closely correlated with PL activities, and negatively related with the changes in flesh firmness. The full-length cDNA of PL genes from the two cultivars was cloned using the RACE method. Amino acid residues of C terminal sequences of the PL proteins from different species showed significant variations. Those results suggested that the differences in PL activity, gene sequence, and gene expression patterns may lead to the different roles of PL in different fruit textures of strawberries during ripening and softening.