Dietary flaxseed (FS) inhibited the growth of human breast tumours and enhanced the effectiveness of tamoxifen (TAM) in athymic mice with low oestradiol (E2) levels. The present study determined whether the n-3 fatty acid-rich cotyledon fraction of FS (FC), alone or in combination with TAM, has a similar effect and thus can substitute for FS. In a 2 × 2 factorial design, ovariectomised mice with established oestrogen receptor (ER)-positive breast tumours (MCF-7) were treated as follows: groups 1 and 2 were fed the basal diet (BD, control) and FC diet (82 g FC/kg), respectively. Groups 3 and 4 with TAM implants (5 mg) were fed the BD and FC diet, respectively. At 8 weeks post-treatment, mice were euthanised, and tumours were analysed by immunohistochemistry and real-time PCR. BD, FC and FC/TAM groups significantly decreased tumour area, but the TAM group did not. Tumour regression in the FC/TAM group was greater compared to the TAM group. FC lowered cell proliferation but had no effect on apoptosis; the opposite was observed with TAM. FC suppressed mRNA expressions of pS2 and insulin-like growth factor 1 receptor (IGF-1R) and protein expressions of ERα, phosphospecific ERα, human epidermal growth factor receptor 2 (HER2), phosphospecific HER2 (pHER2) and amplified in breast 1 (AIB1), while TAM up-regulated mRNA expressions of Bcl2, progesterone receptor and IGF-1R and protein expression of pHER2, and down-regulated ERβ mRNA. FC modulated the effect of TAM on tumour growth biomarkers. In conclusion, FC reduced the growth of ER+human breast tumours at low circulating E2, alone and combined with TAM, in part through modulation of ER − and growth factor-mediated signalling pathways; it may substitute for FS in increasing the effectiveness of TAM.