Abstract: Caspase-9, the most intensively studied caspase, plays a crucial role in mitochondrial or intrinsic apoptosis of mammals as a protease. To explore the role of caspase-9 in fish, we cloned casp9 gene from rainbow trout (Oncorhynchus mykiss) and studied its characteristic and functions. Our in vivo experiments revealed that casp9 expression was significantly upregulated 24 h after bacterial challenge in the gills, accompanied by notable histopathological changes and apoptosis signals. Gene cloning identified that rainbow trout caspase-9 is a 455 amino acid protein encoded by a 1368 bp gene, characterized by a conserved CARD domain, as well as prominent large (P20) and small (P10) subunits. Functional assays of recombinant rainbow trout caspase-9 (rcaspase-9) showed dose-dependent proteolytic activity, with significantly higher activity at 2 mg/mL than at 0.2 mg/mL. Conversely, the CARD domain-deleted version exhibited minimal activity even at 2 mg/mL, demonstrating that the CARD domain is indispensable for homodimerization and self-activation. Additionally, RT-qPCR results indicated that casp9 was mainly expressed in gills and spleen. In vitro studies demonstrated significant upregulation of casp9, apaf-1 and cyt c, along with downregulation of bcl-2 in primary gill cells in response to LPS, peptidoglycan and poly I:C stimulation. Furthermore, rcaspase-9 induced upregulation of casp6 and casp7 in these cells. These findings underscore the importance of caspase-9 in immune defense and apoptosis triggered by pathogens in rainbow trout.