Oral administration of a protein antigen generates a serum factor that induces tolerance when transferred into naïve recipients. This serum factor has been described in rats as consisting of exosome‐like structures or tolerosomes, which express major histocompatibility complex class II molecules (MHCII) and mediate antigen‐specific tolerance. In this study, we investigated the functions of serum‐derived tolerosomes both in vivo and in vitro. Tolerosomes were purified from the 100 000 g pellet fraction of serum from ovalbumin (OVA)‐fed mice. When transferred into naïve recipient mice, the tolerosomes mediated OVA‐specific tolerance. We also found that tolerosomes from OVA‐fed mice induced the activation of OVA‐specific T cells both in vivo and in vitro. The inoculation of severe combined immunodeficiency (SCID) mice with an interferon‐γ‐producing cell line normalized the expression of MHCII in the intestinal epithelial cells and restored their ability to generate tolerosomes. Syngeneic but not allogeneic transfer of tolerosomes from OVA‐fed donors induced tolerance in the recipients. Our results show that tolerosomes can be isolated from mouse serum, that tolerosome‐induced oral tolerance requires MHCII expression in intestinal epithelial cells, and that tolerosomes are functional only in syngeneic recipients.