HER-2 gene amplification or overexpression occurs in approximately 25 % of breast cancers, and trastuzumab (Tmab) is a monoclonal antibody currently used to treat patients with HER-2-overexpressing breast cancer. Signalling through alternative receptor tyrosine kinases, such as insulin-like growth factor I receptor (IGF-IR), has been implicated in resistance to Tmab. The main aim of this study was to investigate mechanisms of resistance and in particular the role of IGF-IR in resistance to Tmab. Response to Tmab was analysed in a panel of HER-2-positive breast cancer cell lines; no correlation was found between HER-2, IGF-IR, EGFR expression or phosphorylation and response to Tmab. However, both HER-2 and phospho-HER-2 levels were found to correlate positively with phospho-IGF-IR levels (HER-2, p = 0.16; p-HER-2, p = 0.002). Tmab (T)-resistant cells showed reduced response to Tmab compared to parental cells. T-resistant BT474 have significantly elevated HER-2, phospho-HER-2, EGFR and phospho-EGFR levels compared to parental cells, while T-resistant SKBR3 cells have significantly elevated IGF-IR levels compared to parental cells (p = 0.026). Targeting IGF-IR with anti-IGF-IR siRNA or an IGF-IR tyrosine kinase inhibitor (TKI) (NVP-AEW541) inhibited the growth of both parental and T-resistant SKBR3 and BT474 cells. Combined treatment with Tmab and IGF-IR inhibitors also inhibited significantly more proliferation than single agents in T-resistant BT474 cells, and in parental and T-resistant SKBR3 cells. SKBR3 cells were conditioned in lapatinib (a dual HER-2/EGFR TKI), and the conditioned SKBR3-L cells showed significantly reduced response to lapatinib, and to Tmab, compared to parental SKBR3 cells. Phosphoproteomic analysis revealed alterations in the levels of a number of phosphoproteins in lapatinib resistant cells. HER-2 and IGF-IR expression were measured by immunohistochemistry in tissue microarrays (TMAs) of patient breast tumour samples. Membrane IGF-IR staining correlated inversely with HER-2 expression (p = 0.026). In conclusion, these data suggest that increased signalling through alternative RTKs may play a role in acquired resistance to Tmab. The combination of anti-IGF-IR therapies with Tmab may be clinically beneficial for patients with HER-2-positive breast cancer. We have identified a number of phosphoproteins with potential involvement in trastuzumab and/or lapatinib resistance, and further analysis of these targets may lead to novel therapeutic targets in HER-2-resistant breast cancer.