We have previously reported that cerebellum modulates immune system function. Since there is no direct structural connection between cerebellum and immune system, exploring pathways mediating cerebellar immunomodulation is important to understand the phenomenon. It has been well known that hypothalamus is a crucial immunoregulatory center, which modulates immune cells via sympathetic system and hypothalamic-pituitary-adrenal axis. Moreover, direct bidirectional connections between the cerebellum and hypothalamus that constitute cerebellar-hypothalamic circuits have been found. Based on these findings, we hypothesized that direct cerebellar-hypothalamic projections transmit cerebellar immunoregulatory information to hypothalamus, through which the regulatory information is then conveyed to immune cells. Here, we focused on cerebellar fastigial nucleus (FN), one of three cerebellar nuclei, to investigate its immunoregulatory pathway via hypothalamus. Sprague-Dawley rats (either sex, 220-240 g) were deeply anesthetized with pentobarbital (55 mg/kg, i.p.) before any surgery and generally recovered to normal state 3-4 h after the anesthesia. Texas red dextran amine (TRDA), a neuroanatomical anterograde tracer, was delivered into FN of rats that had been mounted in a stereotaxic frame following deep anesthesia to observe cerebellar-hypothalamic projections. Fluoro-Ruby (FR), a retrograde tracer, was injected into lateral hypothalamic area (LHA) and 8 days later, the cerebellar sections were stained with immunohistochemistry for GABA. To change cerebellar-hypothalamic GABAergic transmission, we microinjected vigabatrin, an inhibitor of GABA-transaminase that degrades GABA, or 3-mercaptopropionic acid (3-MP), an antagonist of glutamic acid decarboxylase that synthesizes GABA, bilaterally into FN. Immune function, including concanavalin A-induced T cell proliferation, anti-sheep red blood cell (SRBC) IgM antibody and natural killer (NK) cell cytotoxicity, was measured by CFSE/CD3 double labeling, ELISA and flow cytometry, respectively, on day 3 after the drug injection. Simultaneously, GABA content in hypothalamus was tested by HPLC. We found that the TRDA-labeled fibers from FN traveled through superior cerebellar peduncle (SCP), crossed in decussation of SCP, and primarily terminated in LHA. The retrograde tracing with FR from LHA to FN confirmed the direct projection route. Many FR-positive neurons in FN were GABA-immunoreactive. Compared with intact or saline-treated rats, the vigabatrin treatment increased both number of GABA-immunoreactive neurons in FN-LHA projections and GABA content in hypothalamus. Simultaneously, vigabatrin led to a decrease in T cell proliferation, in serum anti-SRBC IgM antibody level and in NK cell cytotoxicity. In support of these findings, 3-MP caused changes that were all opposite to those induced by vigabatrin. These results show that cerebellar FN has a direct GABAergic projection to hypothalamus and suggest that this projection mediates cerebellar immunomodulation.