Omega-3 polyunsaturated fatty acids (n-3 PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are mainly found in marine fish oils and commercially available fish oil supplements. Several studies have documented that n-3 PUFAs can reduce the risk of cardiovascular diseases through anti-inflammatory, antithrombotic, and anti-atherosclerotic properties. Notably, regulation of vascular tone is one of the most important bases of cardiovascular health and especially for maintaining blood pressure within optimal physiological ranges. Recent clinical and animal studies indicate an association between n-3 PUFAs and vascular functions. In this regard, many clinical trials and basic experimental studies have been conducted so far to investigate the influence of n-3 PUFAs on vascular tone. In this review, we have summarized the results obtained from both clinical and basic studies that evaluated the effect of n-3 PUFAs under physiological and pathological conditions. Moreover, we also focus on verifying the underlying basic molecular mechanism of n-3 PUFAs on the vascular system.