PTEN (phosphatase and tension homolog deleted on chromosome 10) has been shown to be inactivated in a wide range of cancers and the role of this gene product is associated with the suppression of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in many cancers. Recently, some reports demonstrated that the degree of PTEN expression could predict trastuzumab chemosensitivity in ErbB2-overexpressing breast cancer. Here, we demonstrate the possible involvement of a proteasome inhibitor (PS341) in PTEN expression and elucidate the influence of PI3K/Akt, one of the main cascades of the ErbB2 downstream pathway, and discuss the role of the proteasome inhibitors in trastuzumab resistance. ErbB2-overexpressing SKBR3 human breast cancer cells and trastuzumab-resistant SKBR3/R cells were analyzed in this study. We show that the expression of phosphorylated Akt was highly increased in trastuzumab-resistant cells, although the expression of PI3K, phosphorylated PI3K and non-phosphorylated Akt was unchanged in comparison with wild-type SKBR3 cells. However, following treatment with PS341, the level of phosphorylated Akt was decreased in a dose-dependent manner. Conversely, the level of PTEN was increased in the same fashion. PS341 showed sufficient cytotoxicity in resistant cells in combination with trastuzumab and the efficacy of trastuzumab was inclined to be better in resistant cells under PS341 treatment. Remarkable activity of Akt was observed in trastuzumab-resistant SKBR3 breast cancer cells and this phenomenon could be associated with the decreased expression of PTEN. The proteasome inhibitor PS341 could increase the level of PTEN and inhibit the downstream pathway of ErbB2, interfering with phosphorylation of Akt.