1H Magnetic Resonance Spectroscopy (MRS), 31P MRS and Diffusion Weighted MRI (DW-MRI) were applied to study the metabolic changes associated with estrogen dependence in estrogen receptor (ER) positive BT-474 and triple negative HCC1806 breast cancer xenografts supplemented with or without 17β-estradiol (E2) at a dose of 0.18 or 0.72 mg/pellet. Furthermore, the effect of estrogen withdrawal on metabolism of BT-474 and HCC1806 breast cancer xenografts were studied on day 0, day 2 and day 10. Increasing the dose of E2 resulted in a rapid growth and increase in lactate level, PME/βNTP, PCr/Pi and βNTP/Pi ratios in BT-474; however no significant changes were found in HCC1806 breast cancer xenografts. Estrogen withdrawal resulted in a marked decrease in lactate level and PME/βNTP and an observed increase in βNTP/Pi, PCr/Pi and apparent diffusion coefficient (ADC) values of BT-474 xenografts on day 10. These data suggest that lactate, PME/βNTP, PCr/Pi and βNTP/Pi ratios of ER-positive tumors were closely related to ER dependence.