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Nowadays, developing countries have seen a reduction in male reproductive parameters, and it has been linked to the exposure of endocrine disrupting chemicals (EDCs), which are able to mimic or disrupt steroid hormone actions. Also, nanoparticles have shown effects on the male reproductive system, in particular the use of TiO 2 -NPs in drugs, cosmetics, and food as pigment additives, and, thanks to their small size (1-100 nm), provide themselves the opportunity to be internalized by the body and pass the blood-testis barrier (BTB). Therefore, TiO 2 -NPs can act on spermatogenesis and spermatozoa. In this study, we carried out an in vitro assay on human spermatozoa to evaluate the effects of TiO 2 -NPs at the concentrations of 500, 250, 100, and 50 ppm. Exposure did not statistically alter sperm parameters (e.g., motility and viability) but induced damage to sperm DNA and the expression of biomarkers by spermatozoa. This immunofluorescence investigation showed a positivity for biomarkers of stress (HSP70 and MTs) on the connecting piece of spermatozoa and also for sex hormone binding globulin (SHBG) biomarkers. The SHBG protein acts as a carrier of androgens and estrogens, regulating their bioavailability; therefore, its expression in the in vitro assay did not rule out the ability of TiO 2 -NPs to act as endocrine disruptors.