Endothelial dysfunction is characterized by decreased vascular NO availability. Elevated NEFA decreases eNOS activity. In cultured cells, we found that fatty acyl CoA synthase (FACS) inhibitor Triacsin C (TC) interrupted eNOS palmitoylation, increasing eNOS activity but not changing vascular‐active eicosanoids. Hypothesis: TC mitigates endothelial dysfunction by increasing NO and decreasing NEFA. In this study, intravascular NO synthesis was measured by electrochemistry in the ischemic hind limb with heparinized rat model in a time course design. The post‐ischemic NO was significantly elevated in TC (100 μg/kg)‐treated animals than controls. NOS inhibitor treatments in this model implicate a role of eNOS, but not iNOS. Acute hyperlipidemia was induced by a bolus dose of Intralipid®(1 ml) to increase NEFA. Pre‐treatment with TC had no effect on total plasma lipids or triglycerides, either before or after Intralipid®. TC reduced baseline plasma NEFA from 240 ± 26.8 to 147 ± 14.3 μg/ml (p=0.038). Fifteen minutes after Intralipid®, NEFA rose to 1461 ± 130 μg/ml (control), which was blunted by TC to 393 ± 2.55 μg/ml (p=0.0079). By 85 min., the difference had subsided (194 ± 10.2 control vs. 281 ± 74.8 μg/ml TC; p=0.74). The TC effect was not uniform across all fatty acid species measured. These data show that TC increases post‐ischemic eNOS activity and blunted plasma NEFA induced by acute hyperlipidemia.