Objective To investigate the regulatory mechanism of Smad signaling pathway mediated by TGF-β1 on extracellular matrix (ECM) of human peritoneal mesothelial cells (HPMCs). Methods The third generation HPMCs from primary culture were divided into the control group and the treatment groups with 5 ng/ml TGF-β1 stimulation. Immunohistochemistry and Western blot assay were used to detect the expression of p-Smad2/3 and its migration in cells. Western blot assay, ELISA and semi-quantification RT-PCR were used to detect the mRNA and protein expressions of Smad7, connective tissue growth factor (CTGF), α- smooth muscle actin (α-SMA), plasminogen activator inhibitor-1(PAI-1), fibronectin (FN) and collagen, type I (COL1). Results ①p-Smad2/3 in HPMCs was also almost not expressed in the control group, and remarkably increased 15 min after TGF-β1 stimulation, peaking at 1 h and dropping after 2h; Meanwhile, p-Smad2/3 mainly distributed in cytoplasm at 15 min, concentrated in cell nucleus and peri-nucleus at 1 h, and distributed in cytoplasm again at 2h; ② The protein expressions of intracellular Smad7, CTGF, α-SMA and COL1 in the treatment groups were more obviously increased than those in the control group, peaking at 48 h such as Smad7, CTGF and α-SMA, and increased in time-dependent manner such as COL1; The protein contents of supernatant PAI-1 in the treatment groups were more significantly increased than that in the control group(24h、48h P<0.01，72h P<0.05), peaking at 24 h, and the protein contents of supernatant FN in the treatment groups were more remarkably increased than that in the control group in a time-dependent manner (P<0.01); The mRNA levels of Smad7 in the treatment groups were more significantly increased than that in the control group in a time-dependent manner (24h P<0.05，48h、72h P<0.01), and the mRNA levels of CTGF、α-SMA、PAI-1、FN and COL1 in the treatment groups were more remarkably increased than those in the control group (P<0.01), peaking at 48 h such as CTGF and α-SMA, peaking at 24 h such as PAI-1, and increased in a time-dependent manner such as FN and COL1. Conclusion TGF-β1 can specifically activate Smad signaling pathway in HPMCs, and participates in the regulatory effects of ECM by inducing the transcription and expression of some downstream target genes such as Smad7、CTGF、α-SMA、PAI-1、FN and COL1 of Smad signaling pathway. 