The myokine irisin can cross the blood brain barrier and act as a neurokine to protect brain function during endurance exercise. However, the mechanism of transport from the blood to cerebrospinal fluid is unknown. Irisin has been detected in rodent and human brain and human cerebrospinal fluid by using commercial antibodies and enzyme linked immunosorbent assay kits. However, as human FNDC5 has an atypical translation start codon, some studies have questioned the specificity of commercial antibodies. Recently, human irisin was identified and quantitated in plasma by using mass spectrometry. We investigated whether there was irisin in human cerebrospinal fluid and an irisin concentration gradient between in human cerebrospinal fluid and paired plasma. An irisin peptide was identified and quantitated by using mass spectrometry with control peptides enriched with heavy stable isotopes as internal standards. Quantitative mass spectrometry identified the presence of irisin in human cerebrospinal fluid. The internal irisin peptides were modified to the deamidated asparagine form after deglycosylation. The unmodified internal irisin peptides were not found in CSF and irisin concentration was approximately 0.26–1.86 ng/ml in men over 80 years of age with various diseases. However, the parallel reaction monitoring (PRM) elution profiles of both modified and unmodified internal irisin peptides were not found in paired plasma samples. These data unequivocally demonstrated the presence of the glycosylated form of irisin in human cerebrospinal fluid. There were significant individual differences in men over 80 years of age with diseases. However, irisin was not detected in plasma samples by using mass spectrometry.