Visceral leishmaniasis is a neglected anthropozoonosis caused by parasitic protozoans from the genus Leishmania, which has the dog as the main urban reservoir in South America and in the Mediterranean basin. Infected dogs may develop neurological signs, ranging from paraparesis to generalized seizures. Nevertheless, we have previously detected that the central nervous system (CNS) of both symptomatic and asymptomatic dogs undergoes inflammatory alterations after natural infection by Leishmania infantum (syn. chagasi). Amastigotes forms of the parasite were not found in the brain, but its DNA was detected by qPCR in different areas of the encephalon, and in situ hybridization allowed us to observe an intense DNA deposition in the choroid plexus, where there was also an up-regulation of toll-like receptors (TLR-2, TLR-9). Furthermore, infected dogs presented remarkable diffuse astroglial and periventricular microglial activation, along with high amount of T lymphocytes infiltration, located especially at the meninges and the choroid plexus. Proinflammatory cytokines (IL-1β, IFN-γ, TNF-α) and chemokines (MIP-1α, MIP-1β, RANTES) were overexpressed, and matrix metalloproteinases enzymes (MMP-2, MMP-9) were detected in the brain and in the cerebrospinal fluid (CSF). These findings suggest a breakdown of the cerebral barriers, which is supported by the presence of anti-Leishmania antibody titers in the CSF along with a negative CSF IgG index. Altogether, these data are further evidence that there is in the brain an inflammatory response during the peripheral infection by this zoonotic protozoan and that the brain should be included in the list of the affected organs during visceral leishmaniasis.