A case of Mycoplasma-associated polyarthritis was diagnosed in a captive reticulated giraffe (Giraffa camelopardalis reticulata). Recurrent episodes of lameness with temporary response to antimicrobial therapy characterized the disease. After the fifth episode, the giraffe was immobilized for arthrocentesis of the right front fetlock joint. Although the culture was negative, Mycoplasma sp. nucleic acid was detected in synovial fluid using polymerase chain reaction (PCR). Twelve weeks after completion of enrofloxacin therapy evidence of Mycoplasma sp. was not detectable in the synovial fluid; no relapses occurred after 22 mo. This is the first report of Mycoplasma-associated polyarthritis in a giraffe.