Titanium　dioxide　nanoparticles　(TiO2-NPs)　have　been　applied　to　a　wide　range　of　applications　in　many　fields　because　of　their　excellent　optical　performance　and　electrical　properties.　Such　broad　applications　increase　their　exposure　to　people.　However,　the　effects　of　exposure　routes　in　long　term　in　vivo　should　be　investigated　in　detail　for　safe　application,　but　are　poorly　understood　to　date.　In　our　study,　we　evaluated　the　effects　of　different　exposure　routes　(single　intravenous,　intragastric,　and　intraperitoneal)　on　the　bio-distribution　and　toxicity　of　different-sized　TiO2-NPs　in　mice.　Our　results　confirmed　that　the　bio-distribution　of　TiO2-NPs　was　dependent　on　the　route　of　exposure,　as　well　as　time　and　size.　The　spleen,　followed　by　the　liver　and　lungs,　had　the　highest　particle　accumulation　for　all　groups　treated　with　TiO2-NPs　of　either　25　nm　or　50　nm　in　size.　Significant　differences　in　the　amount　of　TiO2-NPs　bio-distributed　in　the　different　tissues　were　also　observed　between　the　three　exposure　routes.　The　amount　of　titanium　changed　noticeably　with　time　in　the　main　tissues,　although　at　different　rates.　There　was　also　an　obvious　difference　in　the　coefficients　of　the　organs　between　the　different　exposure　routes.