Neoseiulus californicus (McGregor) is an important predatory mite species characterized by its broad prey spectrum and strong adaptability. ATP-binding cassette (ABC) transporters represent a large protein superfamily present in both prokaryotic and eukaryotic organisms, primarily functioning in xenobiotic transport and maintenance of cellular homeostasis. However, research on this protein family in predatory mites remains largely unknown. In this study a total of 49 putative ABC transporter genes were identified, through a comprehensive analysis of the N. californicus transcriptome. Phylogenetic analysis classified these transporters into eight distinct subfamilies (ABCA-H). Expression profiling of ABCA subfamily genes across different developmental stages demonstrated ubiquitous expression patterns for all members except NcABCA-07. Functional characterization of two highly expressed genes in adult females, NcABCA-02 and NcABCA-05, was performed through RNA interference. Silencing of NcABCA-02 and NcABCA-05 increased the mortality rate of adult female mites from 1.00% to 12.00% and 5.78%, respectively. Furthermore, predation efficiency on adult female Tetranychus urticae Koch significantly decreased from 3.87 prey individuals to 1.87 (51.7% reduction) and 1.70 (56.1% reduction) following silencing of NcABCA-02 and NcABCA-05. Additionally, reduced egg hatching rates were observed in silenced individuals. These results indicate critical roles of NcABCA-02 and NcABCA-05 in maintaining survival, predatory capacity, and reproductive fitness in N. californicus. The findings provide valuable insights into the physiological mechanisms of predatory mites and establish a theoretical framework for enhancing their efficacy in biological control applications.