The epidermal growth factor receptor (EGFR) is frequently overexpressed in various tumours of epidermal origin and is held responsible for tumourigenicity and tumour persistence. Increased nuclear factor (NF)-kappaB activity has been suggested to be involved in the malignant behaviour of EGFR-overexpressing cells. However, the mechanisms that regulate EGF-induced NF-kappaB activation are still largely unknown. Here we show that EGF can induce NF-kappaB-dependent gene expression independently from IkappaBalpha degradation or p100 processing in EGFR-overexpressing HEK293T cells. Moreover, EGF-induced NF-kappaB activation could be inhibited by overexpression of ABINs, which were previously identified as intracellular inhibitors of tumour necrosis factor, interleukin-1 and lipopolysaccharide-induced NF-kappaB activation. Knockdown of ABIN-1 by RNA interference boosted the NF-kappaB response upon EGF stimulation. The C-terminal ubiquitin-binding domain containing region of ABINs was crucial and sufficient for NF-kappaB inhibition. Adenoviral gene transfer of ABINs reduced constitutive NF-kappaB activity as well as the proliferation of EGFR-overexpressing A431 and DU145 human carcinoma cells. Altogether, these results demonstrate an important role for an ABIN-sensitive non-classical NF-kappaB signalling pathway in the proliferation of EGFR-overexpressing tumour cells, and indicate a potential use for ABIN gene therapy in the treatment of cancer.