Currently, trastuzumab resistance is a major clinical problem in the treatment of Her2-overexpressing breast cancer. The underlying molecular mechanisms are not fully understood. Our previous study demonstrates that β2-adrenergic receptor (β2-AR) and Her2 comprise a positive feedback loop in human breast cancer cells and that crosstalk between Her2 and β2-AR affects the bio-behaviors of breast cancer cells, suggesting that the β2-AR activation may be involved in trastuzumab resistance. In this study, we show that the expression of β2-AR, which mediates most catecholamine-induced effects, negatively correlates with trastuzumab response in the patients with Her2-overexpressing breast cancer. Catecholamines potently antagonize the anti-proliferative effects of trastuzumab both in vitro and in vivo. Catecholamine stimulation upregulates the expression of miR-21 and MUC-1 by activating Her2 and STAT3, leading to deficiency of phosphatase and tensin homolog and activation of phosphatidylinositol-3-kinase (PI3K) and Akt. Through inhibition of miR-199a/b-3p, catecholamines induce the mammalian target of rapamycin (mTOR) activation. Thus, trastuzumab resistance-dependent PI3K/Akt/mTOR pathway is controlled by catecholamine-induced β2-AR activation. The data indicate that β2-AR is a reliable molecular marker for prediction of response probability to trastuzumab-based therapy in breast cancer. We also demonstrate that β-blocker propranolol not only enhances the antitumor activities of trastuzumab but also re-sensitizes the resistant cells to trastuzumab. Our retrospective study shows that concurrent treatment of β-blocker and trastuzumab significantly improved progression-free survival and overall survival in the patients with Her2-overexpressing metastatic breast cancer, implicating the possibility for combination therapy with trastuzumab plus β-blocker in Her2-overexpressing breast cancer.