Objective Pituitary adrenocorticotropic hormone (ACTH)-secreting adenoma is a relatively intractable endocrine adenoma that can cause a range of severe metabolic disorders and pathological changes involving multiple systems. Previous studies have shown that celastrol has antitumor effects on a variety of tumor cells via the AKT/mTOR signaling. However, whether celastrol has pronounced antitumor effects on pituitary ACTH-secreting adenoma is unclear. This study aimed to identify a new effective therapeutic drug for pituitary ACTH-secreting adenoma. Methods Mouse pituitary ACTH-secreting adenoma cells (AtT20 cells) were used as an experimental model in vitro and to establish a xenograft tumor model in mice. Cells and animals were administered doses of celastrol at various levels. The effects of celastrol on cell viability, migration, apoptosis and autophagy were then examined. Finally, the potential involvement of AKT/mTOR signaling in celastrol's mechanism was assessed. Results Celastrol inhibited the proliferation and migration of pituitary adenoma cells in a time- and concentration-dependent manner. It blocked AtT20 cells in the G0/G1 phase, and induced apoptosis and autophagy by downregulating the AKT/mTOR signaling pathway. Similar results were obtained in mice. Conclusion Celastrol exerts potent antitumor effects on ACTH-secreting adenoma by downregulating the AKT/mTOR signaling in vitro and in vivo.