We have previously shown that S-nitroso-N-acetylpenicillamine (SNAP) (NO donor) decreased the levels and functions of Giproteins through the formation of peroxynitrite (ONOO^-) in vascular smooth muscle cells (VSMC). The present studies were undertaken to investigate if ONOO^- treatment of VSMC can modulate the expression of Gi protein and associated adenylyl cyclase signaling. Treatment of A10 and aortic VSMC with ONOO^- for 24 hrs decreased the expression of Gi-2 and Gi-3 and not Gs protein in a concentration-dependent manner, which was restored towards control levels by Mn(111)tetralis (benzoic acid porphyrin) (MnTBAP) and uric acid and not by ODQ and KT5823. ONOO^- (0.1 and 0.5 mM) also increased the levels of cGMP by about 50% and 150% respectively which were attenuated toward control levels by 1H[1,2,4]oxadiazole [4,3-a]quinoxaline-1-one (ODQ). In addition, ONOO^- (0.5 mM) attenuated the inhibition of adenylyl cyclase by Angiotensin II (AngII) and C-ANP_4-23 as well as the inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity by GTPS, whereas, the Gs-mediated stimulations were augmented. In addition, ONOO^- treatment (0.5 mM) resulted in decreased phosphorylation of ERK1/2 and P³⁸MAP kinase, while JNK phosphorylation was enhanced and AKT1/3 phosphorylation was not affected. These results suggest that ONOO^- decreased the expression of Gi proteins and associated functions in vascular smooth muscle cells through cGMP-independent mechanism and may involve MAP kinase signaling pathway.