Background Recent genome-wide association studies identified the APOA5/A4/C3/A1 gene cluster polymorphisms influencing triglyceride level and risk of coronary artery disease (CAD). Objectives The purposes of this study were to fine-map triglyceride association signals in the APOA5/A4/C3/A1 gene cluster and then explore the clinical relevance in CAD and potential underlying mechanisms. Methods We resequenced the APOA5/A4/C3/A1 gene cluster in 200 patients with extremely high triglyceride levels (≥10 mm/l) and 200 healthy control subjects who were ethnically matched and genotyped 20 genetic markers among 4,991 participants with Chinese Han ethnicity. Subsequently, 8 risk markers were investigated in 917 early-onset and 1,149 late-onset CAD patients, respectively. The molecular mechanism was explored. Results By resequencing, a number of newly and potentially functional variants were identified, and both the common and rare variants have remarkable cumulative effects on hypertriglyceridemia risk. Of note, gene dosage of rs2266788 demonstrated a robust association with triglyceride level (p = 1.39 × 10-19), modified Gensini scores (p = 1.67 × 10-3), and numbers of vascular lesions in CAD patients (odds ratio: 1.96, 95% confidence interval: 1.31 to 2.14, p = 8.96 × 10-4). Functional study demonstrated that the rs2266788 C allele destroyed microRNA 3201 binding to the 3′ UTR of APOA5, resulting in prolonging the half-life of APOA5 messenger RNA and increasing its expression levels. Conclusions Genetic variants in APOA5/A4/C3/A1 gene cluster play an important role in the regulation of plasma triglyceride levels by an increased APOA5 concentration and contribute to the severity of CAD. © 2014 by the American College of Cardiology Foundation.