Febrifugine, a natural alkaloid, exhibits specific anti-phytophthora activity; however, its mode of action is unclear. In this study, halofuginone, a synthetic derivative of febrifugine, showed significantly higher anti-phytophthora activities than those of febrifugine and the commercial drug metalaxyl against , , and with effective concentration for 50% inhibition (EC) values of 0.665, 0.673, and 0.178 μg/mL, respectively. Proline could alleviate the growth inhibition of halofuginone on , implying that halofuginone might target prolyl-tRNA synthetase (PRS). The anti-phytophthora mechanism of halofuginone was then investigated by molecular docking, fluorescence titration, and enzymatic inhibition assays. The results revealed that halofuginone could bind to PRS and shared a similar binding site with the substrate proline. Point mutations at Glu316 and Arg345 led to 24.5 and 16.1% decreases in the enzymatic activity of PRS but 816.742- and 459.557-fold increases in the resistance to halofuginone, respectively. The results further confirmed that halofuginone was a competitive inhibitor of proline against PRS, and Glu316 and Arg345 played important roles in the binding of halofuginone and proline. Taken together, the results indicated that halofuginone is an alternative anti-phytophthora drug candidate and that PRS represents a potential target for the development of new pesticides.