Vitamin K1 is commonly administered to dairy cattle suffering from uncontrollable hemorrhage and to cattle with known deficiency of vitamin K dependent coagulation factors. However, a review of recent literature concludes the absence of available information regarding the safety and effects of this drug in dairy cattle. Therefore, this study was carried out to evaluate the safety and effects of a single intramuscular injection of vitamin K1 (2.5 mg/kg) on various clinical, hematological, serum biochemical and coagulation parameters in adult Holstein dairy cows. Six adult Holstein dairy cows were injected a single intramuscular dose of vitamin K1. Cows were then clinically monitored for 24 hours after drug administration for any abnormal behavioral activities. The heart rate, respiration rate, rectal temperature, and rumen motility were also reported before and at each follow-up check point after administration of the drug. Whole blood samples were collected before and again at 15, 30, 60, and 120 minutes and at 24 hours after drug administration. Hematology and serum biochemistry parameters were evaluated to detect any systemic effects. Selected coagulation parameters including the activated partial thromboplastine time (APTT), prothrombin time (PT), thrombin time (TT), D-dimers, platelets count, and fibrinogen concentrations were determined to evaluate the effect of the drug on coagulation mechanisms. There were no abnormal clinical, pathological, or behavioral activities associated with the drug administration in all cows. In the coagulation profile, there was a significant increase in platelets counts starting from 15 minutes after administration and throughout the observation period. Other coagulation parameters were not significantly changed.