Background: Calcium-dependent protein kinase (CPK) is one of the main Ca²⁺ combined protein kinase that play significant roles in plant growth, development and response to multiple stresses. Despite an important member of the stress responsive gene family, little is known about the evolutionary history and expression patterns of CPK genes in pineapple. Results: Herein, we identified and characterized 17 AcoCPK genes from pineapple genome, which were unevenly distributed across eight chromosomes. Based on the gene structure and phylogenetic tree analyses, AcoCPKs were divided into four groups with conserved domain. Synteny analysis identified 7 segmental duplication events of AcoCPKs and 5 syntenic blocks of CPK genes between pineapple and Arabidopsis, and 8 between pineapple and rice. Expression pattern of different tissues and development stages suggested that several genes are involved in the functional development of plants. Different expression levels under various abiotic stresses also indicated that the CPK family underwent functional divergence during long-term evolution. AcoCPK1, AcoCPK3 and AcoCPK6, which were repressed by the abiotic stresses, were shown to be function in regulating pathogen resistance. Conclusions: 17 AcoCPK genes from pineapple genome were identified. Our analyses provide an important foundation for understanding the potential roles of AcoCPKs in regulating pineapple response to biotic and abiotic stresses