ABSTRACT Environmental constraints such as drought and salt stress severely limit crop production worldwide. The WRKY TFs are very important in regulating plant growth and stress responses. Pineapple ( Ananas comosus ) is widely grown due to its unique flavor, high vitamins, and dietary fiber; however, the functional characterization of WRKY TFs in pineapple remains largely unexplored. In our study, we amplified and explored the molecular function of pineapple AcWRKY27 , including its conserved domain, protein localization, transcriptional activity, and expression profiles in different tissues and stress treatments. Overexpression of AcWRKY27 in both rice and Arabidopsis thaliana ( A. thaliana ) resulted in growth inhibition, a decrease in primary root elongation, and a reduction in fresh weight under salt, drought, and ABA stress. RNA‐Seq analysis and quantitative PCR (RT‐qPCR) revealed that AcWRKY27 overexpression resulted in decreased expression levels of stress‐responsive and ABA signaling pathway genes. These findings provide new perspectives on pineapple WRKY TFs and lay a foundation for improving pineapple stress tolerance through molecular breeding in the future.