T cell immunoglobulin mucin domain (TIM)-3 is an immunomodulatory molecule and upregulated in T cells by several cytokines. TIM-3 also influences mast cell function but its transcriptional regulation in mast cells has not been clarified. Therefore, we examined the transcript level and the promoter activity of TIM-3 in mast cells. The TIM-3 transcript level was assessed by real-time RT-PCR and promoter activity by luciferase reporter assay. TIM-3 mRNA levels were increased in HMC-1, a human mast cell line by TGF-β1 stimulation but not by stimulation with interferon (IFN)-α, IFN-λ, TNF-α, or IL-10. TIM-3 promoter -349~+144 bp region relative to the transcription start site was crucial for the basal and TGF-β1-induced TIM-3 promoter activities in HMC-1 cells. TIM-3 promoter activity was increased by overexpression of Smad2 and Smad4, downstream molecules of TGF-β1 signaling. Our results localize TIM-3 promoter activity to the region spanning -349 to +144 bp in resting and TGF-β1 stimulated mast cells.