S100A1 is a small Ca²⁺-binding protein, expressed in myocardium and blood vessels, downregulated in diseased heart, and plays a role in regulating cardiac muscle Ca²⁺ homeostasis and contractility. To understand its physiological role under basal conditions and following myocardial infarction (MI), we used a mouse strain with targeted deletion of the S100A1 gene (S100A1 KO). We compared 49 wild-type (WT) and 56 S100A1 KO mice (6-8 weeks old) over 28 days following MI with sham-operated controls. We also examined the effect of S100A1 deficiency on vascular function of isolated blood vessels. S100A1 KO mice demonstrated worse survival compared to WT (21% vs. 69%, respectively, P < 0.001). Hemodynamic evaluation revealed a higher mean arterial pressure (MAP) in KO sham-operated animals compared with WT (99 ± 4 vs. 77 ± 3 mmHg, respectively, P < 0.001), that persisted in both groups post-MI (86 ± 2 vs. 66 ± 4 mmHg, respectively, P < 0.001). Sham-operated male S100A1 KO had higher MAP than female KO mice (122 ± 5 vs. 93 ± 3, respectively P < 0.05) and reduced survival following MI (4% vs. 27%, respectively, P < 0.05). In isolated aortas and mesenteric arteries, acetylcholine-evoked vasodilatation in KO was significantly reduced compared to WT mice (P < 0.05). Nitric oxide production was reduced in endothelial cells isolated from KO. Thus, absence of S100A1 results in acute functional impairment and high mortality following MI associated with a gender-specific hypertensive phenotype. S100A1 appears to play a role in endothelial-dependent regulation of blood pressure.