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Abstract Background Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. An association with neuralgic amyotrophy and Guillain-Barré syndrome (GBS) was previously described. Concerning GBS, studies from other countries found an acute HEV infection in 5–11% of cases. However, HEV prevalence shows considerable regional variations. Therefore, we retrospectively analyzed the frequency of HEV infections in association with GBS in a monocentric cohort in Southwestern Germany. Methods Overall, 163 patients with GBS treated in our clinic between 2008 and 2018 of whom serum and/or cerebrospinal fluid (CSF) samples were available, were identified. Serum samples were analyzed for anti-HEV immunoglobulin (Ig)M and IgG antibodies by ELISA. Additionally, both serum and cerebrospinal fluid (CSF) samples were tested for HEV RNA by PCR if IgM was positive or patients presented within the first 7 days from GBS symptom onset. A group of 167 healthy volunteers and 96 healthy blood donors served as controls. Results An acute HEV infection was found in two GBS patients (1.2%) with anti-HEV IgM and IgG antibodies. HEV PCR in serum and CSF was negative in these two patients as well as in all other tested cases. Seroprevalences indicated that acute infection did not differ significantly from controls (0.8%). Anti-HEV IgG seroprevalence indicating previous infection was unexpectedly high (41%) and revealed an age-dependent increase to more than 50% in patients older than 60 years. Conclusion In this study, serological evidence of an acute HEV infection in patients with GBS was rare and not different from controls. Comparing our data with previous studies, incidence rates show considerable regional variations.