Prolactin-producing tumors represent nearly half of all pituitary tumors. The noticeable rate of aggressive clinical behavior in males with macroprolactinoma necessitates identifying novel genes and proteins which play a role in the process of tumorigenesis, tumor invasion to adjacent structures and resistance to medical treatment. ERα36 expression which is a novel splice variant of traditional ERα66 receptor has not been evaluated in the pituitary gland which is highly responsive to estrogen. In this cohort study, tumor samples from 62 patients with prolactinoma who underwent surgery during a period of eight years were evaluated for immunohistochemistry. ERα36, ERα66, Ki67 and p53 were measured by semi-quantitative immunoreactive score. A wide expression of ERα36 even more than ERα66 was found in normal pituitaries. This may imply the importance of non-genomic signaling pathway of estrogen in the pituitary. The scoring results of Ki67 showed that tumor proliferation rate was higher in males. Males also showed a greater mitotic count than women. Males presented larger and invasive tumors. There were no significant sex-related differences in the expression of the estrogen receptors and p53.