Trihydrophobin 1 (TH1) is a member of the negative elongation factor complex, which is involved in transcriptional pausing. Although the negative elongation factor complex attenuates the estrogen receptor α-mediated transcription, little is known about the relationship between TH1 and tumor progression. Here, we report that the protein level of TH1 was negatively correlated with the aggressiveness of human breast cancer. Immunohistochemical analysis revealed that TH1 expression in clinical stage III–IV primary breast cancer tissues was statistically significantly lower than that in stage I–II breast tissues (< 0.01), and especially inversely associated with lymph node metastasis (< 0.001). Furthermore, we showed that overexpression of TH1 in MDA-MB-231 breast cancer cells inhibited, and knockdown of TH1 in MCF-7 cells enhanced, cell proliferation and migratory ability. Moreover, upregulation of TH1 in MDA-MB-231 cells resulted in the decrease of cyclin D1, β-catenin, and ERK activity, and the increase of p21. In contrast, knockdown of TH1 in MCF-7 cells enhanced the expression of cyclin D1 and β-catenin, increased the activity of ERK, and downregulated the expression of p21. Additionally, overexpression of TH1 in MDA-MB-231 cells prevented. However, knockdown of TH1 in MCF-7 cells induced a number of molecular and cellular alterations associated with epithelial–mesenchymal transition. Taken together, our results suggest that TH1 might play an important role in regulation of proliferation and invasion in human breast cancer, and could be a potential target for human breast cancer treatment. (Cancer Sci 2010); 101: 000–000