Mycoplasma leachii was initially isolated from arthritic calves in South Queensland, Australia, and its ability to cause clinical polyarthritis in calves was demonstrated by experimental infection.  However, the source of M. leachii infection in calves and its means of spreading are not well known.  In this study, one-month-old calves were inoculated with cultures of M. leachii or joint fluid (collected from M. leachii-infected calves) through the intraarticular, intravenous, intratracheal, intranasal or oral routes.  Multidisciplinary procedures, including clinical assessment, etiology assessment, pathology and immunohistochemistry (IHC), were used to evaluate the pathogenicity of M. leachii in calves and to elucidate the transmission route of M. leachii infection in calves.  The results showed that all calves inoculated intraarticularly with cultured GN407 or joint fluid and two-thirds of the calves inoculated intravenously with joint fluid developed severe polyarthritis, and the M. leachii antigen was detected in the joints of all infected calves by IHC and PCR.  In contrast, calves inoculated with cultured M. leachii or joint fluid through the intratracheal, intranasal or oral routes did not show any M. leachii infection in the clinical assessment, etiology assessment, or pathology and IHC results.  These results indicated that polyarthritis caused by M. leachii in calves is transmitted via the blood route; however, this disease is not transmitted through the respiratory or digestive routes.  In addition, the M. leachii antigen was not detected in the lungs of all the inoculated calves using IHC and PCR, indicating that M. leachii is not associated with pneumonia, even in the calves inoculated through the respiratory duct.  These findings are important information for the prevention and control of calf polyarthritis caused by M. leachii.