The nucleus accumbens (NAc), a brain region implicated in the regulation of anxiety-related behaviors, has been found to be susceptible to blood brain barrier (BBB) permeability in psychopathologies. Additionally, the positive effects of testosterone on BBB function and anxiety related behaviours is still controversial. In this study, exploiting natural phenotypic variation in anxiety-like behaviors in outbred Wistar male rats, we aimed at investigating whether testosterone plays a role in anxiety-related BBB disfunction. We classified animals as high (HA) or low anxious (LA) according to the time spent in the open arms of the elevated plus maze. We observed that HA animals exhibit lower levels of testosterone than LA animals. Single nucleus gene expression analyses indicated a significant decrease in the expression levels of several BBB related genes, involving both endothelial and astrocytic markers, in HA animals. Using Evan’s Blue injections, we observed higher blood vessels’ permeability in the NAc of HA animals. Using electron microscopy, we observed a lower number of endfeet processes around blood vessels in the NAc and higher number of discontinuous tight junctions in HA animals. Furthermore, we observed a lower number of contacts between the mitochondria and the endoplasmic reticulum in those endfeets, which is accompanied by a decrease of mitofusin 2 levels. Treating HA animals with physiological levels of testosterone, reversed not only the anxiety phenotype but also ameliorated the molecular BBB disfunctions in these animals. Therefore, our results indicate that testosterone anxiolytics effects act through the modulation of the BBB in male rats.