Summary Head and neck squamous cell carcinoma (HNSCC) is a highly immunosuppressive malignancy. Exosomes in HNSCC patients' plasma are enriched in inhibitory cargo and mediate immunosuppression. As these exosomes are products of various cells, the cellular origin of immunoregulatory proteins they carry is unknown. To test whether tumour‐ or T cell‐derived exosomes in patients' plasma are immunosuppressive and impact upon disease activity, we separated CD3(–) from CD3(+) exosomes by immunocapture using anti‐CD3 antibodies. The exosome protein cargo was evaluated for immunoregulatory proteins using on‐bead flow cytometry. Tumour protein‐enriched CD3(–) exosomes were CD44v3(+). Surprisingly, mean levels of programmed death ligand 1 (PD‐L1), cytotoxic T lymphocyte antigen 4 (CTLA‐4) and cyclooxygenase‐2 (COX‐2) were similar in CD3(+) and CD3(–) exosomes, although the latter induced higher (P < 0·0025) ex‐vivo apoptosis of CD8(+) T cells and greater (P < 0·005) conversion of CD4+ T cells to CD4(+)CD39(+) regulatory T cells (Treg). CD3(+) and CD3(–) exosomes carrying high levels of immunosuppressive proteins were highly effective in mediating these functions. Exosomes of patients with Union for International Cancer Control (UICC) stages III/IV disease had higher levels of PD‐L1 and COX‐2 than stages I/II patients (P < 0·005). Patients with nodal involvement had exosomes with the higher inhibitory protein content than N0 patients (P < 0·03). CD3(+) and CD3(–) exosomes of HNSCC patients had higher PD‐L1, COX‐2 and CD15s levels than healthy donors' exosomes (P < 0·009), although levels of immunostimulatory OX40 or OX40L were not different. By isolating CD3(–)/CD44v3‐enriched and CD3(+) exosomes from plasma, the cellular origins of immunoregulatory proteins they carry were identified. Association of exosome molecular profiles with disease progression supports the exosome potential as future cancer biomarkers. Exosomes from plasma of patients with HNSCC were separated by immunocapture with anti‐CD3 Abs into CD3+ and CD3(‐) fractions. Each fraction was evaluated for the content of immunoregulatory proteins by on‐bead flow cytometry. The levels of immunoregulatory proteins carried by exosomes were correlated to disease activity and stage and lymph node positivity. CD3(‐) CD44v3+ tumor‐derived exosomes were more immunosuppressive than CD3(+) T cell‐derived exosomes.