S-acylation is a crucial post-translational modification that transfers long-chain fatty acids to cysteine residues within proteins, but the fatty acid specificity of this modification in plants remains entirely unknown. In this study, we established a robust system for characterizing the fatty acid specificity of protein S-acylation in plant cells via click chemistry. We applied this approach to identify various S-acylated proteins modified by myristic acid, palmitic acid, or stearic acid in Arabidopsis through chemical proteomics. Additionally, we investigated the preferences for different fatty acid chain lengths among 24 protein S-acyltransferases and examined how elevated temperatures influence the utilization of stearic acid. Functional analyses indicated that specific fatty acids regulate the distribution of protein substrates across distinct membrane regions. Our current research will pave the way for advancements in the field of plant protein S-acylation, providing a powerful tool and valuable resource for future studies involving Arabidopsis and other plant species.