Acinetobacter baumannii (A. baumannii) is an important pathogen causing various nosocomial infections. CRISPR-Cas system is the adaptive immune system of bacteria, which is also closely related to the drug resistance and virulence of bacteria. However, the effect and mechanism of cas3 (type I-Fa) in A. baumannii is still unclear. In this study, we successfully constructed a cas3 deletion mutant (19606Δcas3) and complemented strain (19606Δcas3/pcas3) to study the regulatory mechanism of type I-Fa cas3 on bacterial virulence. Our results showed that deletion of cas3(type I-Fa) significantly reduced the biofilm formation, virulence and pathogenicity to mice. The organ bacterial load of mice infected with cas3 deletion strain was significantly reduced, the lung inflammation was slightly changed, and the serum cytokine level was also decreased. All results demonstrated that cas3 enhanced the virulence and pathogenicity of A. baumannii. Mechanism analysis showed that deletion of cas3 can lead to the down-regulation of virulence factors such as biofilm formation related factors and outer membrane protein A(ompA). In addition, cas3 was also involved in the regulation of carbon metabolism and oxidative phosphorylation pathway of A. baumannii. Altogether, our study may provide cas3 as a therapeutic target in the future because of the close link to the virulence of A. baumannii.