Abstract Body: Epitranscriptomic modifications to RNA provide a critical layer of control for RNA processing, stability, and functional regulation; specifically, due to altered interactions with receptors and RNA-binding proteins. Small non-coding RNAs (sRNA) likely retain these post-transcriptional modifications (PTxM) from their parent transcripts of which they were cleaved. Although the biological functions of each PTxM are unknown, evidence supports that PTxMs likely regulate innate immune responses and inflammation. As HDL transport sRNA fragments from classes of RNA known to harbor extensive PTxMs, we predicted that HDL likely harbor regulatory PTxMs that may contribute to HDL functionality in disease. We posit that increased PTxM levels on HDL-sRNA cargo in ASCVD contribute to newly observed, direct effects of HDL particles towards immune cells in the setting of ASCVD. To assess HDL-PTxM levels in ASCVD, total RNA were isolated from HDL particles of healthy individuals and ASCVD patients with coronary artery calcium (CAC) scores >21 Agatston units. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and AlkB-facilitated RNA (de)methylation sequencing (ARM-seq), we quantified HDL-sRNA-PTxMs. LC-MS/MS revealed a significant increase in modified nucleosides in ASCVD. ARM-seq identified enriched tRNA-derived fragments (tDRs) in CAC+ individuals compared to CAC-, including tDR-ArgACG-1-1 harboring 1-methyladenosine (m1A) modifications. To quantify direct effects of ASCVD-HDL towards macrophages, primary macrophages were treated with CAC+ or control HDL without prior inflammatory stimuli. Bulk RNA (mRNA) sequencing identified many altered genes in pathways related to calcium signaling, myeloid differentiation, and cell adhesion, including increased expression of TMEM123, a transmembrane protein implicated in immune cell migration and cytoskeletal organization. Furthermore, recombinant HDL particles loaded with m1A-tDR-ArgACG-1 significantly increased TMEM123 mRNA and protein levels in treated macrophages. These findings suggest that HDL-delivered m1A-modified tDRs influence immune signaling and macrophage activation, promoting pro-inflammatory phenotypes within atherosclerotic lesions. This study identifies HDL-associated RNA modifications as novel modulators of immune responses, with therapeutic potential for targeting modified HDL-tDRs in cardiovascular disease.