Identification of new binding targets is essential for the development of herbicides and phytotoxin-tolerant crops. Caprylic acid (CAP) is a safe and non-selective bio-herbicide in uncultivated areas. However, the herbicidal action of CAP remains unclear. Herein, metabolomic and proteomic profiling indicated that a serine hydroxymethyl transferase inConyza canadensis(CcSHMT1) is a promising candidate binding targeted for CAP. The protein abundance and activity ofCcSHMT1 were decreased in a time- and dosage-dependent manners after CAP treatment. CAP competes with phenyl-serine at the binding sites, decreasing the enzymatic activity ofCcSHMT1. Overexpression ofCcSHMT1, AtSHMT1andOsSHMT1inArabidopsisor rice endowed plants with high tolerance to CAP treatment, whereas the knockout ofosshmt1led to death of plants under normal atmospheric conditions. Furthermore, T194A, T194A/ M195V and D209NOsshmt1mutant plants derived from base editing exhibited tolerance to CAP. CAP bound toCcSHMT1 with high affinity, and Ala191 in the key domains of N-terminus was identified to be critical for the binding site of CAP. Collectively, our findings demonstrate thatCcSHMT1 is a binding target for herbicidal activity of CAP. This study marks a key step in the druggability of SHMT inhibitors and represents an attractive target for phytotoxin-tolerant crops.