Acute aortic dissection (AAD) is an acute inflammatory vascular condition associated with significant morbidity and mortality. Depletion of neutrophils can attenuate the development of AAD. The CXC-motif chemokine 5 (CXCL5) can attract and activate neutrophils. This study aimed to investigate whether direct inhibition of CXCL5 could protect against AAD formation. A set of AAD animal models was designed using an angiotensin II infusion for 3 days after treatment with the lysyl oxidase inhibitor beta-aminopropionitrile for 4 weeks in 4-week-old male BALB/c mice. While AAD developed successfully in all the animals, approximately 31% of the mice died before sacrifice. The morphological changes at different time points during the experimental period indicated that angiotensin II could trigger AAD formation in this model. CXCL5 protein expression in the aorta tissue was increased after treatment with angiotensin II. Moreover, the ex vivo and in vitro study showed that vascular smooth muscle cells and monocytes isolated from the animals could generate CXCL5. CXCL5 inhibition by a specific monoclonal antibody significantly decreased the severity of AAD evaluated by ultrasound, aorta wet weight, and en face assay. The immunohistochemical analysis showed that the aortic tissues from AAD mice had higher expressions of matrix metalloproteinase (MMP) 9 and neutrophil-positive areas in the medial layer compared to control mice. Treatment with a CXCL5 antibody reduced MMP9 and neutrophil expressions as well as neutrophil and CXCL5 double-positive areas compared to untreated AAD mice. In conclusion, direct inhibition on CXCL5 reduced aortic MMP9 expression as well as neutrophil infiltration and attenuated the development of AAD, suggesting the mechanistic role of CXCL5 in neutrophil-triggered AAD. CXCL5 may be a potential therapeutic target for AAD.