Little is known about changes in calpain activity in the pituitary gland. In the present study, mu- and m-calpain activity changes were detected in the rat anterior pituitary following intraperitoneal injection of diethylstilbestrol. Double-immunofluorescence labeling confirmed colocalization of mu- and m-calpain in prolactin-secreting cells (lactotrophs). Western blot analysis revealed significantly increased expression of both calpains, which accompanied upregulated cytosol and membrane zymographic activities at 12 weeks following diethylstilbestrol injection, compared with rats injected with sunflower oil. Moreover, following estrogen injection, pituitary gland pathological damage gradually worsened with increasing time. Results demonstrated that estrogen regulated calpain expression and activity, and both calpains participated in the pathophysiological processes of the pituitary gland. Ubiquitous calpain expression could serve as an effective target for anti-estrogen drugs.