Mitochondrial DNA: A new driver for sex differences in spontaneous hypertension.  The prevalence of arterial hypertension (AH) is higher in men than in premenopausal women of the same age. AH has been characterized as a chronic inflammatory disease and activation of Toll-like receptors (TLR) by damage-associated molecular patterns (DAMPs) is involved. Mitochondrial DNA (mtDNA) may be released by end-organ damage, which is recognized and activates TLR9. The serum level of mtDNA is increased in AH. The aim of this study was to compare the serum mtDNA levels between male and female spontaneously hypertensive rats (SHR) and to evaluate the sex differences in the effect of mtDNA on the function, inflammation and signaling pathway related to TLR9 in the vasculature. Male and female 15-week-old SHR and Wistar rats were used to evaluate the arterial blood pressure, serum mtDNA, contractile response, inflammatory markers and signaling pathway related to TLR9. Male SHR had higher arterial blood pressure values and serum mtDNA compared to female SHR and to male and female normotensive Wistar rats. In male SHR aorta, mtDNA incubation increased the contractile response to phenylephrine, which was blunted by inhibition of TLR9, and also increased pro-inflammatory molecules IL-6 and TNF-α. However, in female SHR aorta, mtDNA incubation did not change the contractile response, reduced pro-inflammatory molecules and prevented oxidative stress. mtDNA incubation did not change the expression of TLR9, MyD88 and eNOS neither in male nor in female SHR aorta, but it increased the phosphorylation of ERK1/2 in male and reduced in female SHR aorta. The mtDNA differential modulation of vascular response in male and female SHR might contribute to sex differences in AH. This study contributes to the understanding of a need for more personalized therapeutic strategies for men and women with hypertension. Keywords: Sex differences, Arterial hypertension, Mitochondrial DNA, Toll-Like receptor 9.