<p>Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) act as critical post-transcriptional regulators in the pathogenesis of atherosclerosis (As). However, the non-canonical regulatory mechanisms of lncRNAs and miRNAs, independent of the competing endogenous RNA (ceRNA) pathway, especially in homocysteine (Hcy)-induced vascular smooth muscle cells (VSMCs) dysfunction—a key event in As progression—remain largely unclear. In the present study, high-throughput small RNA sequencing, combined with bioinformatics analysis, identified miR-212-5p as a pivotal mediator of Hcy-induced VSMCs hyperproliferation, increased migration, and decreased apoptosis. Both in vitro and in vivo experiments consistently demonstrated that Hcy significantly upregulated the expression of lncRNA-H19 and miR-212-5p in VSMCs and aortic media of hyperhomocysteinemic (HHcy) ApoE⁻/⁻ mice. Mechanistically, combined with gain- and loss-of-function assays, dual-luciferase reporter assays and molecular interaction experiments, our results suggest that lncRNA-H19 can directly interact with miR-212-5p to form a regulatory-associated molecular cascade, which jointly downregulates Krüppel-like factor 4 (KLF4) expression. Notably, this interaction mode may differ from the canonical ceRNA sponge regulatory mechanism and may represent a potential non-canonical interaction pattern. Further functional experiments indicate that homocysteine modulates the expression of the lncRNA-H19/miR-212-5p axis to reduce KLF4 levels, and is involved in mediating homocysteine-triggered vascular smooth muscle cell dysfunction. In summary, the present study preliminarily suggests that a potential regulatory association that may not depend on the classical ceRNA mechanism may exist between the lncRNA-H19/miR-212-5p axis and KLF4 in homocysteine-related atherosclerosis. We speculate that these two molecules may serve as potential candidate biomarkers and research-oriented intervention targets for hyperhomocysteinemia-associated atherosclerosis.</p>