Background Metformin decreases serum levels of monomeric prolactin. No previous study has investigated the effect of metformin on macroprolactin content in patients with macroprolactinemia. Methods We studied three age-, sex- and weight-matched groups of patients: 15 women with monomeric prolactin, 12 women with macroprolactin, as well as 15 women with normal prolactin levels. Because of coexisting 2 diabetes or prediabetes all patients were treated with metformin (1.7-3 g daily). Plasma lipids, glucose homeostasis markers, as well as serum levels of prolactin and macroprolactin were assessed at baseline and after 4 months of metformin treatment. Results As expected, metformin reduced plasma glucose and triglycerides, as well as improved insulin sensitivity in all treatment groups. Moreover, the drug reduced post-polyethylene glycol prolactin levels and tended to reduce pre-polyethylene glycol prolactin levels in women with monomeric prolactin but not in women with macroprolactinemia and women with normal prolactin levels. Conclusion The obtained results indicate that metformin has a negligible effect on macroprolactin levels.