We aimed to evaluate the effects of fulvestrant on the glycolysis of prolactinoma GH3 cells, and reveal the potential regulatory mechanisms. Prolactinoma cell line GH3 was treated with different concentrations of fulvestrant (0, 0.12, 0.25, 0.5 and 1 ng/ml) for 4 h. siRNAs XBP1s and XBP1u were constructed to treat GH3 cells. The expression levels of XBP1s, XBP1u, IRE1, PKM2 and GRP78 of GH3 cells were detected by Western blot. Meanwhile, the glycolytic activity of GH3 cells, including the glucose uptake, ATP/ADP, and lactate production were detected. The expression levels of XBP1s and XBP1u were significantly inhibited by fulvestrant in a dose-dependent manner. The glucose uptake, ATP/ADP and lactate production of GH3 cells were significantly inhibited by fulvestrant as well as siRNA XBP1s and XBP1u (p < 0.05). Western blot analysis suggested that the expression levels of IRE1, PKM2 and GRP78 were significantly decreased in GH3 cells treated by fulvestrant as well as siRNA XBP1s and XBP1u, compared with those in normal control (p < 0.05). Fulvestrant could inhibit the glycolysis of GH3 cells by downregulating IRE1/XBP1 signaling pathway, and this process was closely related with the downregulation of PKM2.