Experimental studies suggest that n-3 polyunsaturated fatty acids (n-3 PUFA) may have the potential to modulate mammary gland development and reduce breast cancer (BC) risk. Experimental studies provide evidence that n-3 PUFA can inhibit mammary tumourigenesis in rodents, however, findings from epidemiological studies are inconsistent. Direct evidence to support an anti-cancer role for n-3 PUFA is needed to address this controversy. Therefore, this study used novel transgenic mouse models to investigate the influence of lifelong n-3 PUFA exposure on mammary tumourigenesis. Heterozygous transgenic ' fat-1' mice, which can endogenously produce n-3 PUFA, were crossed with heterozygous MMTV-neu(ndl)-YD5 mice, an aggressive BC model that over-expresses human epidermal growth factor receptor-2 (HER-2/neu). The novel double-hybrid mouse produced provides a direct means with which to investigate the effects of lifelong n-3 PUFA exposure on mammary tumour development. Mice expressing MMTV-neu(ndl)-YD5 and 'fat-1' exhibited significant (p<0.01) reductions in mammary tumour volume and multiplicity over time, compared to mice only expressing MMTV-neu(ndl)-YD5. These findings suggest an important role for maternal nutrition and lifelong intake of n-3 PUFA in BC prevention.