M10, a spiroplasma isolated from honeybee showed a helical morphology and a contractile movement in R-2 medium. The isolate M10 could pass through a 220nm membrane and resist to penicillin (2000U/ml). It must grow in medium with serum. Glucose could be used as its source of carbon instead of sucrose. Arginine could be metabolized and urea could not. The 16S rDNA of M10 shared 99.86% similarity with the typical strain Spiroplasma melliferum BC-3 (＝ATCC33219). M10 can cause serious disease to honeybees through feeding cultures, the honeybees showed symptoms of spiroplasmosis around 4 days after feeding and the death rate reached to 71% around 15 days. The isolation rate of spiroplasmas from dead honeybees was as high as 100%. Using primers of Spiroplasma-specific 16S rDNA，16S rDNA of spiroplasma was amplified in the various parts of honeybees, such as heart, chest, abdomen and appendage of the dead honeybees. The results indicated that M10, which caused spiroplasmosis, could infect honeybees systemically.