Cluster of differentiation (CD) 69 is a leukocyte activation receptor involved in the maintenance of immune homeostasis and is positively selected in activated regulatory T (T_reg) cells, implicating its role during T_reg‐cell differentiation. By RNA interference, we show that CD69 is not sufficient to support the conversion of CD4⁺ naive T cells into T_reg cells, whereas it does that of human peripheral blood mononuclear cells (hPBMCs) (P < 0.01), suggesting that a ligand‐receptor interaction is required for CD69 function. Using immunoprecipitation and mass spectrometry, we identified the S100A8/S100A9 complex as the natural ligand of CD69 in hPBMCs. CD69 specifically associates with S100A8/S100A9 complex as confirmed by in vitro binding and competition assay, and the treatment of CD69 with peptide‐N‐glycosidase significantly abolishes such association. In agreement, the glycomics analysis determines the glycosylation site and the N‐glycan composition of CD69, and terminal removal of sialic acid from that N‐linked glycans reverses the generation of forkhead box P3‐positive T_reg cells (23.21%; P < 0.05). More specifically, we showed that CD69‐S100A8/S100A9 association is required for the up‐regulation of suppressor of cytokine signaling 3 resulting in inhibited signaling of signal transducer and activator of transcription 3 (36.54% increase upon CD69 silencing; P< 0.01). This might in turn support the secretion of key regulator TGF‐β (∼3.28‐fold decrease upon CD69 silencing; P < 0.05), leading to reduced production of IL‐4 in hPBMCs. Our results demonstrate the functional and mechanistic interplays between CD69 and S100A8/S100A9 in supporting T_reg‐cell differentiation.—Lin, C.‐R., Wei, T.‐Y. W., Tsai, H.‐Y., Wu, Y.‐T., Wu, P.‐Y., Chen, S.‐T. Glycosylation‐dependent interaction between CD69 and S100A8/S100A9 complex is required for regulatory T‐cell differentiation. FASEB J. 29, 5006–5017 (2015). www.fasebj.org