Ts65Dn mice have been developed as a model for Down syndrome (DS). Because of its involvement in complex behaviors, including sexual and aggressive behaviors, we investigated the nitric oxide (NO) system in specific brain regions of these mutant mice (TS) after isolation-induced aggression. Male TS mice displayed significantly higher aggression than wild type (WT) mice and the comparison of the NO system, both with immunohistochemical and histochemical methods, resulted in robust differences between TS and WT mice in the hypothalamic paraventricular nucleus, in the nucleus of the diagonal band and in the medial septum, but not in the striatum of TS mice. In conclusion, we document alterations in the neuronal NO system of the TS mouse model of DS, suggesting a correlation of the behavioral aggressiveness with deficient NO production.