Cardiovascular diseases, primarily caused by atherosclerosis, are a major public health concern worldwide. Atherosclerosis is characterized by chronic inflammation and lipid accumulation in the arterial wall, leading to plaque formation. In this process, macrophages play a crucial role by ingesting lipids and transforming into foam cells, which contribute to plaque instability and cardiovascular events. Recent studies have suggested that various pathogens are involved in the development of atherosclerosis, with Mycoplasma pneumoniae considered one of the potential candidates. Therefore, this study investigated whether this bacterium induces lipid accumulation in macrophages, which play a crucial role in the development of atherosclerosis, using the Raw264.7 model. Our findings revealed that M. pneumoniae infection promotes lipid droplet formation in macrophages. Glycerol 3-phosphate oxidase, GlpD, in the bacterium is involved in this process by producing reactive oxygen species, which in turn causes the oxidation of low-density lipoprotein. Furthermore, the significant increase in the expression of oxidized lipid receptors involved in the uptake of this oxidized lipid indicates that the bacteria promote lipid uptake in infected macrophages. These results suggest that M. pneumoniae has a direct pro-atherogenic effect, promoting the formation of atherosclerotic lesions through foam cell formation. Understanding the mechanisms by which M. pneumoniae influences atherosclerosis provides valuable insights for devising new therapeutic strategies for the prevention and management of cardiovascular diseases.