Antibodies to certain self-antigens are detected in normal individuals as well as in patients with autoimmune diseases. Natural autoantibodies found in normal human sera are thought to act as an immune regulator, a functional controller of specific proteins, or the first-line defense against pathogenic microorganisms. In the course of screening human pancreatic islet cDNA library with human sera, we found that autoantibodies to galectin-9 and its unique isoform are present in normal healthy individuals. Galectin-9 antibody was detected in all 44 human sera tested by the immunoprecipitation assays, suggesting a widespread presence of galectin-9 autoantibodies in humans. The reactivity of the sera to galectin-9 was not inhibited by lactose or endoglycosidase treatment, indicating that the reactivity was not due to a nonspecific lectin-carbohydrate interaction. We also demonstrated by RT-PCR that galectin-9 and its isoform are expressed in a variety of human tissues such as pancreatic islets, liver, lung, and tonsils as well as HeLa and Jurkat cell lines. Thus, although it has been reported previously that human galectin-9 is expressed mainly in immune cells and tissues, the current work suggests that the expression of galectin-9 and its isoform is not tissue-restricted and natural autoantibodies against them are present in normal human sera. The significance of these autoantibodies needs to be studied further.