Abstract Background Information regarding measurement and supplementation of vitamin K1 (vitK1) in dogs with chronic enteropathy (CE) is limited. Hypothesis/Objectives Compare vitK1 concentrations of healthy dogs to dogs with CE and determine if supplementation with vitK1 increases vitK1 concentrations compared to placebo. Animals Twenty client-owned dogs with CE and 20 healthy university-owned research colony dogs. Methods Prospective, randomized, placebo-controlled study. Dogs with CE were randomly assigned to receive placebo or vitk1 2.5 mg/kg PO q12h for 3 weeks. Vitamin K concentrations were measured pre- and post supplementation using liquid chromatography tandem mass spectrometry and compared to vitK1 concentrations in the healthy cohort. Results All healthy dogs had initial vitK1 median concentrations of 0.10 ng/mL (interquartile range [IQR], 0.05), which was similar to dogs that received either placebo (n = 5; 0.10 ng/mL; IQR, 0.05) or vitK1 (n = 7; 0.10 ng/mL; IQR, 0.05) before supplementation. Dogs with CE receiving vitK1 had increased vitK1 concentrations (12.5 ng/mL; IQR, 4.1) after 3 weeks of supplementation compared with baseline (0.10 ng/mL; p < 0.001), placebo group after 3 weeks (0.10 ng/mL; p < 0.0001) and healthy dogs (0.10 ng/mL; p < 0.004). Conclusions and Clinical Importance Oral supplementation with vitK1 increased vitK1 concentration in the serum of dogs with CE, but a clinical benefit from increased vitK1 concentrations was not identified. The absence of difference in vitK1 concentrations between healthy and CE dogs before supplementation requires additional investigation.