A physiological membrane-receptor agonist typically stimulates oscillations, of varying frequencies, in cytosolic Ca²⁺ concentration ([Ca²⁺]_i). Whether and how [Ca²⁺]_i oscillation frequency regulates agonist-stimulated downstream events, such as gene expression, in non-excitable cells remain unknown. By precisely manipulating [Ca²⁺]_i oscillation frequency in histamine-stimulated vascular endothelial cells (ECs), we demonstrate that the gene expression of vascular cell adhesion molecule 1 (VCAM1) critically depends on [Ca²⁺]_i oscillation frequency in the presence, as well as the absence, of histamine stimulation. However, histamine stimulation enhanced the efficiency of [Ca²⁺]_i-oscillation-frequency-regulated VCAM1 gene expression, versus [Ca²⁺]_i oscillations alone in the absence of histamine stimulation. Furthermore, a [Ca²⁺]_i oscillation frequency previously observed to be the mean frequency in histamine-stimulated ECs was found to optimize VCAM1 mRNA expression. All the above effects were abolished or attenuated by blocking histamine-stimulated generation of intracellular reactive oxygen species (ROS), another intracellular signaling pathway, and were restored by supplementary application of a low level of H₂O₂. Endogenous NF-κB activity is similarly regulated by [Ca²⁺]_i oscillation frequency, as well as its co-operation with ROS during histamine stimulation. This study shows that [Ca²⁺]_i oscillation frequency cooperates with ROS to efficiently regulate agonist-stimulated gene expression, and provides a novel and general strategy for studying [Ca²⁺]_i signal kinetics in agonist-stimulated downstream events.