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Members of the 14-3-3 family are intracellular dimeric phosphoserine-binding proteins that can associate with and modulate the activities of many proteins. In our efforts to isolate the genes regulated by progesterone (P 4 ) using suppressive subtractive hybridization, we previously found that 14-3-3t is one of the genes upregulated by P 4 . In this study, we demonstrated by quantitative RT-PCR (qRT-PCR), western blot analyses, and immunohistochemistry that 14-3-3t mRNA and protein levels were increased in the rat uterus after P 4 treatment. Furthermore, qRT-PCR indicated that P 4 increased 14-3-3t mRNA levels in human endometrial epithelial cells and endometrial stromal cells (ESCs). Western blot and qRT-PCR analyses revealed that in vitro decidualization using cAMP and medroxyprogesterone 17-acetate increased levels of 14-3-3t mRNA and protein in ESCs. We have shown by qRT-PCR and western blot analyses that P 4 increased the mRNA and protein levels of 14-3-3t in Ishikawa cells that stably express P 4 receptor-B (PR-B). Immunocytochemistry revealed that 14-3-3t colocalizes with PR and translocates from the cytoplasm to the nucleus in response to P 4 . Moreover, by luciferase reporter assay, we demonstrated that 14-3-3t enhances the transcriptional activity of PR-B. Taken together, we propose that 14-3-3t is a P 4 -responsive gene in uterine cells that modulates P 4 signaling.