The expression and functional significance of NADPH oxidase 5 (Nox5) and its five isoforms in vascular cells is poorly understood. The goal of this study was to determine whether Nox5-α, -β, -δ, -γ, and -ε (short) are expressed in human blood vessels and evaluate their respective functions. Nox5 mRNA and protein were detected in human blood vessels, cultured human vascular smooth muscle (HVSMC) and endothelium, but not fibroblasts. The most abundant isoforms were α and β, whereas δ and γ were not detected. Nox5-α and -β produced reactive oxygen species (ROS), but -δ, -γ, and -ε were not catalytically active. Coexpression of the active Nox5 isoforms with inactive Nox5 variants suppressed ROS production, and coimmunoprecipitation revealed that Nox5-β binds the inactive ε variant, which may account for reduced ROS production. In HVSMC, angiotensin II, endothelin-1 and TNF-α increased endogenous Nox5 mRNA levels, while adenovirus-mediated overexpression of Nox5 promoted p38 MAPK, JAK2, JNK, and ERK1/2 phosphorylation in endothelial cells (EC), but only increased ERK1/2 phosphorylation in HVSMC. At higher levels of Nox5, there was evidence of increased apoptosis in EC, but not in HVSMC, as detected by the presence of cleaved caspase-3 and cleaved poly(ADP-ribose)polymerase. Although catalytically inactive, Nox5-ε potently activated ERK in HVSMC, and increased expression of Nox5-ε promoted HVSMC proliferation. Nox5 is expressed in human blood vessels. The Nox5-α and -β splice variants are the major isoforms that are expressed and the only variants capable of ROS production. Nox5-ε can inhibit Nox5 activity and activate ERK and HVSMC proliferation.