The liver kinase B1 (LKB1) is encoded by the STK11 gene and acts as a tumour suppressor and a regulator of energy homeostasis. LKB1 expression is reduced in primary breast tumours compared to normal breast epithelium. Although its expression in primary tumours does not appear to correlate with estrogen receptor (ER) status, it is differentially expressed in breast cancer cell lines where ER-negative cells have lower LKB1 expression than ER-positive cells. The present study aimed to examine the effects of estradiol on LKB1 expression and activity in the ER-positive breast cancer cell line MCF-7. Results demonstrate that estradiol causes a dose-dependent decrease in LKB1 transcript and protein expression and consistent with this, a significant decrease in the phosphorylation of the LKB1 target AMPK (P ≤ 0.05). In order to assess whether effects of estradiol were due to effects on ERα binding to the STK11 promoter, ChIP was performed. Results demonstrate that ERα binds to the STK11 promoter in a ligand-independent manner and that this interaction is decreased in the presence of estradiol. Moreover, STK11 promoter activity is significantly decreased in the presence of estradiol (P ≤ 0.05). LKB1 transcript and IHC score were assessed in primary tumours of 18 patients and demonstrated no significant correlation with ER status (n = 18). Our results thereby provide a mechanism whereby LKB1 is decreased in ER-positive breast tumours.