MiR-29b has been reported to function as a tumor suppressor in a variety of cancers. However, its role in the regulation of breast cancer is controversial. In this paper, we explored the expression of miR-29b in a cohort of 67 pairs of formalin-fixed paraffin-embedded specimens with detailed pathological and clinical characteristics, and further analyzed the effects of miR-29b on the malignant phenotype of HER-2-positive breast cancer cells and the relevant mechanisms involved. We found that the miR-29b expression is negatively associated with HER-2 expression in breast cancer tissues. Moreover, overexpression of miR-29b induced a complex phenotype in HER-2-positive breast cancer cells, namely an inhibition of cell proliferation, block of G1/S phase transition, induction of cell apoptosis, suppression of cell invasion in vitro, as well as inhibition on tumor growth in vivo, indicating that miR-29b functions as a tumor suppressor in HER2-positive breast cancer cells. Further bioinformatic prediction suggested that oncogene Stat3, which is an up-stream regulator of HER-2, was a target gene of miR-29b in breast cancer cells. We have shown that knocking down of Stat3 attenuated the malignant phenotype of breast cancer cells similar to overexpression of miR-29b, while restore expression of Stat3 in HER-2-positive breast cancer cells partially abolished the suppressive effects of miR-29b. Collectively, our data suggest that miR-29b could reverse the malignant phenotype of HER-2-positvie breast cancer through, at least partially, targeting Stat3 signaling pathway.