The aim of the current study was to investigate the cytotoxic effects of hypotonic (iopamidol) and isotonic (iodixanol) contract media (CMs) in vitro and in vivo. A total of 60 Wistar rats were included and were randomly divided into three groups (20 rats per group). Iodixanol (4 g iodine/kg), iopamidol (4 g iodine/kg) or equal volume of normal saline was injected via tail vein. HUVEC and H5V cell viability was determined by Cell Counting Kit-8 agents. Western blotting was performed to detect ATP-binding cassette subfamily G member 1 (ABCG1) expression. For histological analysis, hematoxylin and eosin staining was performed. Plasma endothelin, von Willebrand factor, tissue type plasminogen activator, plasminogen activator inhibitor, D-Dimer, fibrinogen, anti-thrombin III, plasminogen and nitric oxide synthase (NOS) were measured by using ELISA. Both iopamidol and iodixanol treatments deceased cell viability and increased apoptosis of HUVEC and H5V cells, along with downregulated NOS and ABCG1. The injection of iopamidol or iodixanol into rats changed the endothelium-related plasma levels of biomarkers, including endothelin, von Willebrand factor, tissue type plasminogen activator, plasminogen activator inhibitor, D-Dimer, fibrinogen and anti-thrombin III. However, endothelia isolated from rat abdominal aorta in the iodixanol group retained their normal structure, whereas endothelial structure in the iopamidol group was injured and disrupted. The findings in the present study suggested that both hypotonic and isotonic CMs may lead to endothelial dysfunction and thrombin and fibrinolytic system disorder. However, hypotonic CMs may be more toxic than isotonic CMs. Therefore, additional cautions should be taken when selecting hypotonic CMs and their dosages during cardioangiography.