A 36-year-old man with no history of immunosuppression presented with diabetes insipidus, but no other hormonal disturbances. Magnetic resonance imaging of the brain revealed an enhanced mass in the pituitary stalk appearing as a thickened pituitary stalk. The mass lesion was completely removed through the right optico-carotid space. Histological examination showed epithelioid cell granuloma with caseous necrosis, which strongly suggested mycobacterial infection. However, acid-fast staining detected no bacteria. Polymerase chain reaction (PCR) examinations of the gastric juice and cerebrospinal fluid for tuberculosis were negative. Nested PCR and deoxyribonucleic acid (DNA) sequencing of the DNA from the surgical specimen disclosed Mycobacterium tokaiense DNA sequences. This rare case of pituitary stalk granuloma caused by M. tokaiense shows that if the surgical specimen contains caseous necrosis, nested PCR and DNA sequencing are useful methods to identify mycobacterial infection.