Author summary The development and evaluation of new treatment strategies againstBabesiaandTheileriaparasites have become increasingly urgent due to the emergence of parasite resistance and unwanted toxicity side effects by current chemotherapies. On the other hand, vaccination is a cheaper, reliable and sustainable option. Unfortunately, it has not worked well for the protozoan diseases because they possess ingenious mechanisms to evade the host immune system, rendering treatment the most suitable approach for their control. Sadly, only diminazene aceturate (DA) and imidocarb dipropionate have passed clinical trials for the treatment of piroplasmosis. However, these drugs cause many adverse effects and not approved yet for human medicine. Cryptolepine (CRY) is a pharmacologically active indoloquinoline alkaloid isolated from the roots of the shrubCryptolepis sanguinolenta. CRY is reported to possess various pharmacological activities, including antifungal, anti-mycobacterial, and potent antiplasmodial activities. In the present study we evaluated the effects of CRY against the growth ofBabesia bovis,B.bigemina,B.divergens,B.caballiandTheileria equi in vitroand its chemotherapeutic potential onBabesia microtiin mice. Furthermore, we investigated the effect of combination between CRY with the current babesiocidal drugs such as DA, atovaquone (AQ) and clofazimine (CF)in vitroandin vivo.Piroplasmosis treatment has been based on the use of imidocarb dipropionate or diminazene aceturate (DA), however, their toxic effects. Therefore, the discovery of new drug molecules and targets is urgently needed. Cryptolepine (CRY) is a pharmacologically active plant alkaloid; it has significant potential as an antiprotozoal and antibacterial under differentin vitroandin vivoconditions. The fluorescence assay was used for evaluating the inhibitory effect of CRY on fourBabesiaspecies andTheileria equi in vitro, and on the multiplication ofB.microtiin mice. The toxicity assay was evaluated on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half-maximal inhibitory concentration (IC50) values of CRY onBabesia bovis,B.bigemina,B.divergens,B.caballi, andT.equiwere 1740 +/- 0.377, 1400 +/- 0.6, 790 +/- 0.32, 600 +/- 0.53, and 730 +/- 0.025 nM, respectively. The toxicity assay on MDBK, NIH/3T3, and HFF cell lines showed that CRY affected the viability of cells with a half-maximum effective concentration (EC50) of 86.67 +/- 4.43, 95.29 +/- 2.7, and higher than 100 mu M, respectively. In mice experiments, CRY at a concentration of 5 mg/kg effectively inhibited the growth ofB.microti, while CRY-atovaquone (AQ) and CRY-DA combinations showed higher chemotherapeutic effects than CRY alone. Our results showed that CRY has the potential to be an alternative remedy for treating piroplasmosis.