Atherosclerosis is the most common cardiovascular disease, leading to complications such as myocardial infarction and stroke, the main but not the only causes of which are lipid accumulation and inflammation. In this study, we investigated whether opioid receptor blockade impacts factors involved in atherosclerosis development and examined the expression of the Hmmr gene as a potential new factor playing a role in the development of this disease. We administered naloxone (NLX) to 8-week-old and 36-week-old ApoE-/- mice, then examined the expression of Hmmr, Col1a1, and Col3a1 in the aorta, as well as the influence of NLX administration on aortic collagen layer thickness and proteomic changes in the aorta. Additionally, we assessed the impact of NLX on the splenic T cell populations. The results showed reduced Hmmr expression in the aortas of both 8-week-old and 36-week-old mice. Col1a1 expression remained unchanged, while col3a1 expression decreased in young mice but increased in older mice. In 36-week-old mice, NLX administration led to an increase in aortic collagen layer thickness. HMMR expression on T cells increased in 36-week-old mice but remained unchanged in 8-week-old mice. Proteomic analysis identified 587 proteins that were altered following NLX treatment. Our studies suggest that both the opioid system and the Hmmr gene are important factors in atherosclerosis development.