The successive events of shedding and regulated intramembrane proteolysis are known to comprise a fundamental biological process of type I and II membrane proteins (e.g. amyloid precursor protein, Notch receptor and pro‐tumor necrosis factor‐α). Some of the resulting fragments were shown to be involved in important intra‐ and extracellular signalling events. Although shedding of the human transferrin receptor‐1 (TfR1) has been known for > 30 years and soluble TfR1 is an accepted diagnostic marker, the fate of the remaining N‐terminal fragment (NTF) remains unknown. In the present study, we demonstrate for the first time that TfR1‐NTF is subject to regulated intramembrane proteolysis and, using MALDI‐TOF‐TOF‐MS, we have identified the cleavage site as being located C‐terminal from Gly‐84. We showed that the resulting C‐terminal peptide is extracellularly released after regulated intramembrane proteolysis and it was detected as a monomer with an internal disulfide bridge. We further identified signal peptide peptidase‐like 2a and mainly signal peptide peptidase‐like 2b as being responsible for the intramembrane proteolysis of TfR1‐NTF. Shedding and regulated intramembrane proteolysis (RIP) are fundamental biological processes of membrane proteins. The resulting fragments are involved in important intra‐ and extracellular signaling events. We demonstrate that the transferrin receptor is subject to RIP and identified the protease SPPL2b being responsible for cleavage at Gly‐84. The resulting C‐peptide is extracellularly released as a monomer with an internal disulfide bridge