Length and aspect ratio represent important toxicity determinants of fibrous nanomaterials. We have previously shown that anatase TiO2nanofibers (TiO2 NF) cause a dose-dependent decrease of cell viability as well as the loss of epithelial barrier integrity in polarized airway cell monolayers. Herein we have investigated the impact of fiber shortening, obtained by ball-milling, on the biological effects of TiO2 NF. Long TiO2NF (L-TiO2 NF) were more cytotoxic than their shortened counterparts (S-TiO2 NF) towards alveolar A549 cells and bronchial 16HBE cells. Moreover, L-TiO2 NF affected the trans-epithelial electrical resistance of 16HBE monolayers. This effect was associated with altered distribution of tight-junction proteins and also mitigated by fiber shortening. Macrophages efficiently internalized S-TiO2 NF but not L-TiO2 NF, which caused cell stretching and deformation. In macrophages S-TiO2 NF enhanced the expression of pro-inflammatory genes, NO production and cytokine secretion, which was significantly inhibited by the phagocytosis inhibitor cytochalasin D. In vivo experiments indicated length-dependent toxicity in both the lungs and peritoneal cavity of mice, leading to significant increase in markers of inflammation in animals treated with L-TiO2 NF. It is concluded that fiber shortening mitigates NF detrimental effects on cell viability and epithelial barrier competence. As far as inflammation is concerned, shortening enhances phagocytosis and macrophage activation in vitro but prevents the increase of inflammatory cytokines upon in vivo exposure. These data suggest that fiber shortening may represent an effective safe-by-design strategy for mitigating TiO2 NF toxic effects both in vitro and in vivo.