Objective The cerebellum has been well known as a structure of subcortical motor control.Herein, we provide new evidence for cerebellar immunomodulation and pathway mediating the modulation.Methods The cerebellar interposed nuclei (IN), one of three cerebellar nuclei were bilaterally lesioned with kainic acid (KA) injections.Functions of T, B and natural killer cells were measured by MTT, ELISA and flow cytometry, respectively.An anterograde tracing with dextran-texas red (Dex-TR) from the cerebellar IN to the hypothalamus was used to demonstrate a direct cerebellohypothalamic projection.A retrograde tracing with fluoro-ruby from the hypothalamus to the cerebellar IN combined with immunofluorescence of glutamate in the IN was employed to determine glutamatergic cerebellohypothalamic projections.Results (1) On days 8, 16 and 32 after KA injection in the IN of rats, lymphocyte percentage in peripheral leukocytes, concanavalin A-induced lymphocyte proliferation, serum sheep red blood cell-specific IgM antibody and natural killer cell toxicity were significantly attenuated with respect to IN-saline control rats.(2) On a series of brain coronal sections from the cerebellum (injected with Dex-TR in the IN) to the hypothalamus, we observed that Dex-TR-labeled nerve fibers originating from cerebellar IN neurons mostly terminated in lateral hypothalamic area (LHA).The retrograde tracing from the LHA to the cerebellar IN and immunofluorescence of glutamate on cerebellar sections showed that the cerebellohypothalamic projections had glutamatergic fibers.(3) Reduction of the cerebellohypothalamic glutamatergic projections by injection of glutaminase inhibitor 6-diazo-5-oxo-l-norleucine in the bilateral cerebellar IN resulted in significant attenuation of both lymphocyte number and proliferation, which was consistent with those caused by the cerebellar IN lesions with KA.Conclusion The cerebellum participates in modulation of immune function and this effect is mediated by cerebellohypothalamic gtutamatergic projections.