Herpes simplex encephalitis (HSE), targeting the limbic system, is the most common cause of viral encephalitis in the Western world. Two pathways for viral entry to the central nervous system (CNS) in HSE have been suggested: either via the trigeminal nerve or via the olfactory tract. This question remains unsettled, and studies of viral spread between the two brain hemispheres are scarce. Here, we investigated the olfactory infection as a model of infection and tropism of herpes simplex virus 1 (HSV-1), the causative agent of HSE, in the CNS of rats. Rats were instilled with HSV-1 in the right nostril and sacrificed 1-6 days post-infection, and tissues were analysed for viral spread using immunohistochemistry and quantitative PCR (qPCR). After nasal instillation, HSV-1 infected mitral cells of the olfactory bulb (OB) on the right side only, followed by limbic encephalitis. As a novel finding, the anterior commissure (AC) conveyed a rapid transmission of virus between the right and the left OB, acting as a shortcut also between the olfactory cortices. The neuronal cell population that conveyed the viral infection via the AC was positive for the water channel protein aquaporin 9 (AQP9) by immunohistochemistry. Quantification of AQP9 in cerebrospinal fluid samples of HSE patients showed increment as compared to controls. We conclude that the olfactory route and the AC are important for the spread of HSV-1 within the olfactory/limbic system of rats and furthermore, we suggest that AQP9 is involved in viral tropism and pathogenesis of HSE.