The serotonergic system is one of the major systems targeted in the pharmacological treatment of mood disorders including depression. Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been reported to induce the expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of serotonin. The 14-3-3 protein family not only activates neuronal enzymes, including TPH, but also plays a role in a wide variety of cell signaling. The aim of the present study was to determine whether fluoxetine regulates both the interaction of TPH and 14-3-3 proteins as well as the increase of those proteins in the dorsal raphe nucleus and the hippocampus. Sprague-Dawley rats were administered fluoxetine or vehicle for 5 and 14 days and sacrificed at 5 and 14 days after initial treatment. The intensity of immunoreactivity for TPH and 14-3-3 proteins in the dorsal raphe nucleus of the midbrain and in the hippocampus was measured, and the colocalization of both proteins was observed with double-labeling immunofluorescence. At 5 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein increased in both the dorsal raphe nucleus and the hippocampus, while that of TPH did not change in either region. In addition, at 14 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein significantly increased in both the dorsal raphe nucleus and hippocampus, while that of TPH showed few changes in either region. Colocalization of TPH and 14-3-3 proteins was observed in the cell bodies of dorsal raphe nucleus, whereas it was not observed in the hippocampus. These results suggest that the time-dependent regulation of 14-3-3 protein may be one of the various factors associated with delayed pharmacological effects of SSRIs.