Sulfatide (3′sulfogalactosylceramide) is a glycosphingolipid present within the nervous system and in the islets of Langerhans. Anti-sulfatide antibodies have been observed in both pre-diabetic and newly diagnosed type 1 diabetic patients. The aim of this study was to test in vivo, the therapeutic effect of sulfatide on the development of diabetes in the NOD mouse. In four separate experiments diabetogenic splenocytes from newly diabetic NOD mice were injected iv into 7-8 week old irradiated (700R) female NOD mice (4-10 million cells/mouse). Each experiment consisted of four treatment groups to which the mice were randomly divided: 1) sulfatide; 2) galactosylceramide (the precursor to sulfatide without sulfate); 3) GM1, a glycosphingolipid negatively charged as sulfatide but with a different sugar composition; and 4) phosphate buffered saline (PBS). The mice received 100 μg glycosphingolipid iv on the day of cell transfer and 1-3 times thereafter at four day intervals, and were screened for diabetes three times a week the next 52 days. Among all the 35 sulfatide-treated mice 54% became diabetic compared to 93 % of 43 PBS-treated animals (p < 0.00001). Correspondingly, galactosylceramide reduced diabetes incidence to 52% (25 mice, p < 0.00001). On the other hand, 86% of GM1-treated mice (n=28) became diabetic indicating that no effect was obtained by this glycosphingolipid. In two experiments in which less spleen cells were transferred (4-5 mill.) and glycosphingolipids were given 4 times, 35% of the sulfatide-treated animals (n = 17) developed diabetes compared to 85% of PBS-treated mice (n = 20, p < 0.001). A robust proliferative response to sulfatide, but none to GM1, was observed when spleen cells were rechallenged with glycosphingolipid in vitro. Thus, like insulin and GAD, sulfatide is able to prevent diabetes in NOD mice