Updated project metadata. We evaluated the anti-leishmanial efficacy of different saturated medium-chain fatty acids (FAs, C8-C18) where FA containing C8 chain, caprylic acid (CA), was found to be most potent against Leishmania donovani, the causative agent for visceral leishmaniasis (VL). Different analogs of CA with C8 chain, but not higher, along with a carboxyl/ester group showed a similar anti-leishmanial effect. Ergosterol depletion, measured by HPLC, was the major cause of CA-mediated cell death. Molecular docking and Molecular dynamic simulation studies indicated the enzyme mevalonate kinase (MevK) of the ergosterol biosynthesis pathway as a possible target of CA. Enzyme assays with purified recombinant MevK and CA/ CA analogs confirmed the target with a competitive inhibition pattern. The inhibition constant of CA was ⁓53µM. Using Isothermal calorimetry and circular dichroism studies; strong binding interaction between MevK and CA/CA analogs was established. Point mutation with Y167F residue at MevK active site reduces the binding affinity of CA on MevK. Also, homologous over-expression of MevK in parasites resisted CA-mediated killing. Further, proteomics studies using LC-HRMS showed dose-dependent down-regulation of MevK in CA-treated cells. We established the mechanism of the antileishmanial effect of CA, a natural product, against VL where toxicity and drug resistance with currents chemotherapeutics demand an alternative. This is the first report on the identification of an enzymatic target with kinetic parameters & mechanistic insights against any organism for a natural medium-chain FA. CA and, even, its derivatives/analogs may be developed as an anti-parasitic formulation with higher efficacy and reduced toxicity.