Background <p>High levels of circulating interleukin (IL)-16 are associated with a reduced incidence of cardiovascular events. The disruption of atherosclerotic plaques commonly causes myocardial infarction and stroke. In this study, we investigated the effects of IL-16 on phenotypic modification of plaques.</p> Methods <p>Mice with deficiencies in IL-16 and apolipoprotein E (IL16^−/−ApoE^−/−) were generated and used for plaque phenotype analysis. RNA sequencing was performed to identify changes in cellular processes and molecular expression in response to IL-16 deficiency.</p> Results <p>IL-16 deficiency increased the necrotic core and reduced fibrous cap thickness in the plaques. IL-16 deletion accelerated the degradation of intraplaque collagen and elastin, increased matrixmetalloproteinase activity, and reduced TIMP-3 expression. Transplantation of wild-type IL-16 bone marrow into IL-16 knockout mice successfully attenuated the plaque instability caused by IL16 deficiency. Furthermore, hematopoietic-derived IL-16 activated the CD4/JAK2/STAT6 pathway and increased the binding of STAT6 to the coactivator cAMP-response element-binding protein (CBP)/p300 at the TIMP-3 promoter in smooth muscle cells (SMCs). Consequently, acetylation of STAT6 and histone H3 increased more than 2-fold, which caused a 2.2-fold upregulation of TIMP-3. Moreover, the anti-atherosclerotic effects of IL-16 on plaque stability were abrogated by the SMC-specific deletion of CD4, and the plaque vulnerability caused by IL-16 defects was reversed by SMC-specific overexpression of TIMP-3.</p> Conclusions <p>IL-16/CD4/JAK2/STAT6 upregulates TIMP-3 expression in SMCs to remodel the intraplaque extracellular matrix toward a stable phenotype. Our findings suggest that IL-16 is a novel factor in vascular remodeling and atherosclerotic plaque phenotype modulation and is a potential target for intervention in the later stages of atherosclerosis.</p>