We report a case of an intravascular hemolytic reaction attributable to anti-Jk b antibodies that were not detected using an enzyme phase antibody identification test. A 61-year-old male who had received two units of red blood cells was admitted to the emergency room because his urine was dark. LISS/Coombs gel column agglutination tests suggested the presence of anti-Jk b and anti-E antibodies. However, his serum was negative for the Jk b antigen when an enzyme phase test was performed. A positive reaction was evident, however, when EDTA-treated plasma was tested; this excluded any possible complement-mediated reaction. The patient was diagnosed with an intravascular hemolytic transfusion reaction, caused by anti-Jk b , and was later discharged without specific complications after receiving antigen-negative blood transfusions. (Korean J Blood Transfus 2016;27:79-83)