Vitamin K1 (VK1) has been widely used as a coumarin antagonist and for the treatment of hemorrhagic disease in veterinary practice. However, the potential mechanism of adverse reaction after VK1 injection has been not fully elucidated. In this study, two cases of anaphylactic reactions after subcutaneous VK1 injection were presented, and then an experimental study was performed to further characterize the anaphylactic reactions. Two dogs developed anaphylactic reactions after subcutaneous VK1 injections and were promptly treated with antihistamines and glucocorticoids, after which abnormal signs related to anaphylaxis disappeared. Subsequently, a study was undertaken to ascertain the nature of the adverse reactions to subcutaneous VK1 injection. Six healthy laboratory beagle dogs received subcutaneous VK1 administrations once daily for eight days. They were monitored for clinical signs after each injection, and blood samples were collected for the measurement of plasma histamine and immunoglobulin E concentrations using enzyme-linked immunosorbent assay. All six dogs showed mild angioedema after the VK1 injections. The dogs also displayed clinical signs including sneezing, coughing, skin reddening, excess salivation, pawing the ground, and somnolence on days 4, 6, and 8. Plasma histamine and immunoglobulin E concentrations were significantly increased by the repeated injections. In summary, this study describes anaphylactic reactions resulting from subcutaneous VK1 administration in dogs. Clinicians should be aware that the repeated subcutaneous injection of VK1 can trigger an anaphylactic reaction in dogs.