Babesia parasites are responsible for enormous economic losses to the livestock industry worldwide. There is no vaccine available to protect from the infection. RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we evaluated the effects of RNAi by double-stranded RNA (dsRNA) of the DNA gyrase subunits A and B, the DNA-directed RNA polymerase beta subunit (rpo B), and thiostrepton interaction site (ribosomal L11 protein) genes on the in vitro growth of Babesia bovis. The inhibitory effects produced by dsRNAs of DNA gyrase subunit A, DNA gyrase subunit B, thiostrepton interaction site (L11) and the DNA-directed RNA polymerase beta subunit (rpo B1and B2) were compared with the effect of drugs target these genes. RNAi directed towards these genes resulted in inhibition of in vitro growth of the parasite. The RT-PCR showed that parasites treated with dsRNAs of DNA gyrase subunit A, DNA gyrase subunit B, ribosomal L11, and the DNA-directed RNA polymerase beta subunit (rpo B1 and B2) have blocked expression of corresponding endogenous mRNAs of DNA gyrase subunit A, DNA gyrase B, ribosomal L11, and rpo B genes. These results demonstrate that the RNAi technique disrupted gene function in B. bovis and the mechanism of action required further studies to be elucidated.