Autoimmune hypophysitis is a rare but increasingly recognized disease of the pituitary gland. Its autoantigens are unknown and the management difficult because it is often misdiagnosed as a non-secreting adenoma. By immunizing female SJL/J mice with mouse pituitary extracts, we established a new mouse model of experimental autoimmune hypophysitis (EAH). Immunized mice developed severe lymphocytic infiltration in the anterior pituitary that closely mimicked the human pathology. In the early phase of EAH, the pituitary enlarged, consistent with the compression symptoms reported by hypophysitis patients at presentation. In the florid phase, adrenal insufficiency and pituitary antibodies developed, in strong correlation with the pituitary pathology. In the late phase, hypothyroidism ensued and the pituitary gland became atrophic. Using immune sera as probes in a 2D-immunoblotting screen followed by mass spectrometry, we identified several proteins that could function as pituitary autoantigens. These findings provide new insights into the pathogenesis of autoimmune hypophysitis and establish a platform for developing novel diagnostic biomarkers and therapeutics.