Pythium insidiosum is the etiological agent of pythiosis, a chronic granulomatous disease affecting people and several animal species, mainly horses. Immunotherapy has recently arisen as a promising therapeutic alternative for the disease. This article reports a preliminary characterization of the specificity of the humoral response to P. insidiosum antigens. The immunodominant P. insidiosum proteins were identified by western immunoblot using sera from naturally infected horses and cattle, and sera from rabbits immunized with mycelial mass antigen (MMA). Sera from naturally infected horses and from immunized rabbits reacted with five main polypeptides of 33.5-, 38-, 39-, 40-, and 80-kDa, respectively. Sera from horses submitted to immunotherapy with MMA showed the same pattern of reactivity. Sera from two naturally infected calves reacted with four main polypeptides: two of them presented a molecular mass of 39-and 40-kDa and were probably the same as those recognized by horse and rabbit sera, whereas the other two (35-and 70-kDa) were exclusively recognized by cattle serum. The initial characterization of P. insidiosum immunodominant antigens may help in understanding the immune response behind the clinical cure obtained through immunotherapy and in designing more effective immunotherapeutic strategies.