The gene encoding the receptor tyrosine kinase ERBB2, also known as HER2, is amplified and/or overexpressed in up to 15% of breast cancers. These tumours are characterised by an aggressive phenotype and poor clinical outcome. Although therapies targeted at ERBB2 have proven effective, many patients fail to respond to treatment or become resistant and the reasons for this are still largely unknown. Using a high-throughput functional screen we assessed whether genes found to be recurrently amplified and overexpressed in ERBB2+ve breast cancers mediate resistance to the ERBB2-targeted agent lapatinib. Lapatinib-resistant ERBB2-amplified breast cancer cell lines were screened, in the presence or absence of lapatinib, with an RNA interference library targeting 369 genes recurrently amplified and overexpressed in both ERBB2-amplified breast cancer tumours and cell lines. Small interfering RNAs targeting a number of genes caused sensitivity to lapatinib in this context. The mechanisms of resistance conferred by the identified genes were further investigated and in the case of NIBP (TRAPPC9), lapatinib resistance was found to be mediated through NF-κB signalling. Our results indicate that specific amplified and/ or overexpressed genes found in ERBB2-amplified breast cancer may mediate response to ERBB2-targeting agents.