Disclosed is crystalline levoisovaleryl spiramycin III and corresponding preparations, preparing methods and uses thereof. The preparations comprise crystalline levoisovalerylspiramycin III and pharmaceutically acceptable carrier and/or adjuvant, wherein the purity of levoisovalerylspiramycin III is above 90 wt%. The crystalline levoisovalerylspiramycin III has a good antibacterial activity, and the preparations include solution for injection, powder for injection or lyophilized powder for injection.