Thrombosis　occurs　in　both　macrovasculature　and　microvasculature,　causing　various　cardio-cerebral　vascular　diseases.　The　lack　of　effective　and　safe　antithrombotic　drugs　leads　to　a　public　health　crisis.　Mounting　evidence　suggests　that　protein　disulfide　isomerase　(PDI)　plays　a　critical　role　in　the　initial　stage　of　thrombus　formation,　motivating　the　research　of　the　feasibility　of　PDI　inhibitors　as　novel　anti-thrombotics.　Rutin,　one　of　the　most　potent　PDI　inhibitors,　was　reported　to　suppress　platelet　aggregation　and　thrombosis　in　animal　models,　but　further　studies　and　clinical　translation　were　restricted　due　to　its　low　aqueous　solubility　and　oral　bioavailability.　In　this　work,　we　fabricated　rutin-loaded　lipid-based　nano-formulation　(NanoR)　and　characterized　their　physical-chemical　properties,　release　profiles,　pharmacokinetic　process,　and　pharmacodynamic　function　against　thrombosis　in　macrovessels　and　microvessels.　NanoR　provided　increased　solubility　and　dissolution　of　rutin　to　achieve　earlier　T　(max)　and　higher　C　(max)　than　the　sodium　salt　of　rutin　(NaR)　after　oral　gavage.　Ex　vivo　studies　demonstrated　that　NanoR　significantly　inhibited　thrombin　generation　and　clot　formation　in　the　plasma　of　mice.　Importantly,　such　effect　was　reversed　by　exogenous　recombinant　PDI,　demonstrating　the　specificity　of　the　NanoR.　In　direct　current-induced　arterial　thrombosis　model　and　ferric　chloride-induced　microvascular　thrombosis　model,　NanoR　exhibited　greatly　enhanced　antithrombotic　activity　compared　with　NaR.　NanoR　also　showed　good　safety　performance　according　to　tail　bleeding　assay,　global　coagulation　tests,　and　histological　analysis.　Overall,　our　current　results　indicated　that　NanoR　offers　a　promising　antithrombotic　treatment　with　potential　for　clinical　translation.