MAPK signal transduction pathways are important regulators of stress responses, cellular growth, and differentiation. In Neurospora, the circadian clock controls rhythms in phosphorylation of the p38-like MAPK (OS-2); however, the mechanism for this regulation is not known. We show that the WCC, a transcription factor and clock component, binds to the os-4 MAPKKK promoter in response to light and rhythmically in constant darkness, peaking in the subjective morning. Deletion of the WCC binding sites in the os-4 promoter disrupts both os-4 mRNA and OS-2 phosphorylation rhythms. The clock also indirectly regulates rhythmic expression of the histidyl-phosphotransferase gene, hpt-1, which peaks in the evening. Anti-phase expression of positive (OS-4) and negative (HPT-1) MAPK pathway regulators likely coordinate to enhance rhythmic MAPK activation to prepare cells to respond to osmotic stress during the day in the natural environment. Consistent with this idea, we show that wild type cells have a clock-dependent morning kinetic advantage in glycerol accumulation after salt stress as compared to evening treatment. Thus, circadian transcriptional control of MAPK pathway components leads to striking time-of-day-specific effects on the signaling status and physiological response of the pathway.