Reactive oxygen species participate in regulating intracellular signaling pathways. Herein, we investigated the reported opposite effects of hydrogen peroxide (H 2 O 2 ) on metabolic signaling mediated by activated a 1 -and b-adrenoceptors (ARs) in hepatocytes. In isolated rat hepatocytes, stimulation of a 1 -AR increases H 2 O 2 production via NADPH oxidase 2 (NOX2) activation. We find that the H 2 O 2 thus produced is essential for a 1 -AR-mediated activation of the classical hepatic glycogenolytic, gluconeogenic, and ureagenic responses. However, H 2 O 2 inhibits b-AR-mediated activation of these metabolic responses. We show that H 2 O 2 mediates its effects on a 1 -AR and b-AR by permeating cells through aquaporin 8 (AQP8) channels and promoting Ca 2+ mobilization. Thus, our findings reveal a novel NOX2-H 2 O 2 -AQP8-Ca 2+ signaling cascade acting downstream of a 1 -AR in hepatocytes, which, by negatively regulating b-AR signaling, establishes negative crosstalk between the two pathways.