Regulation of T cells is necessary to limit their proliferation and to prevent autoimmune diseases. High expression of the cluster of differentiation protein 52 (CD52) results in suppression of other T cells by interacting with sialic acid-binding immunoglobulin-like lectin receptor-10 (Siglec-10) (1). CD52 is a low molecular weight (1208 Da) glycopeptide with a single N-linked, and several potential O-linked, glycosylation sites (2). We aimed to perform a comprehensive glycomics/glycoproteomics characterisation of several CD52-Fc recombinant proteins to determine the bioactive glycoforms of CD52. CD52 was expressed as a recombinant protein in fusion with carrier immunoglobulin Fc glycoprotein in host cells (HEK293 or CHO), and was functionally tested for suppression of T-cell proliferation and interferon-γ secretion. N-glycans were released after PNGase F treatment and reduced glycans were analysed using porous graphitised carbon-liquid chromatography-MS/MS in (-) mode on an ion trap mass spectrometer. Intact-mass of CD52 analysis was performed by (+) mode reversed phase C8-ESI-MS/MS on a Q-TOF mass spectrometer. The N-glycosylation profile of CD52 with the carrier Fc N297 glycosylation site mutated showed the presence of several bi-, tri- and tetra-antennary sialylated N-glycan structures. This glycan profile was also observed on CD52 after Factor X cleavage from the Fc carrier. O-glycan structures were observed by intact-mass analysis of the CD52 glycopeptide after PNGase F treatment, and showed a low abundance of core type-2 di-silaylated structures. Interestingly, the relative abundance of tri- and tetra- antennary sialylated structures, as well as the specific α-2, 3 sialic acid linkage, correlated with two variants of CD52-Fc that showed different immunosuppressive activity. Furthermore, anion exchange chromatography on a Mono Q GL column separated CD52-Fc into glycoforms that varied in their capacity to suppress T-cell proliferation, strongly supporting different sialylation as a determinant of active CD52. These findings define glycan structures responsible for the bioactivity of CD52.