Anti-HER2 antibody trastuzumab is emerging as a frontline therapy for patients with metastatic breast cancers that overexpress HER2. Understanding the molecular mechanisms by which the antibody inhibits tumor growth should permit the design of even more effective trastuzumab-based protocols. Several groups including our own have demonstrated that induction of cyclin-dependent kinase (CDK) inhibitor p27Kip1 protein is one of the key mechanisms of action of HER2-targeting antibodies. In this review, we discuss currently available data regarding the multiple signaling targets and pathways by which HER2-targeting antibodies upregulate p27Kip1 protein in breast cancer cells that overexpress HER2. Anti-HER2 antibodies inhibit HER2-mediated signaling in cancer cells, ultimately upregulating the levels and activity of p27Kip1 protein. At least six signaling targets and pathways are modulated by trastuzumab. By inhibiting CDK2 and decreasing Thr187 phosphorylation of p27Kip1, trastuzumab abrogates targeting of SCF-ubiquitin E3 ligase and minimizes proteasome degradation of p27Kip1. By inhibiting AKT and human kinase interacting stathmin (hKIS), trastuzumab blocks Thr157-, Thr198- and Ser10-induced p27Kip1 translocation from the nucleus to the cytosol, which increases the inhibitory effect of p27Kip1. By inhibiting Jun activation domain-binding protein 1 (Jab1) trastuzumab increases nuclear retention of p27Kip1. By inhibiting cyclin D and c-Myc, trastuzumab releases the sequestrated p27bKip1 protein from cyclin D-CDK4/6 complexes and increase the effect of p27Kip1 on CDK2-cyclin E complexes. By stimulating minibrain related kinase (MIRK), trastuzumab stabilizes p27Kip1 in the nucleus, which increases inhibitory action of p27Kip1 on CDK2. The targets and pathways affected by trastuzumab work in concert to maximize the expression and inhibitory effect of p27Kip1, which leads to cell cycle G1 arrest and growth inhibition.