Updated publication reference for PubMed record(s): 31113380. Manganese (Mn), an essential element for plants, can be toxic when present in excess. Stylo (Stylosanthes) is a pioneer tropical legume with great potential for Mn tolerance, but its Mn tolerance mechanisms remain poorly understood. In this study, the mechanisms underlying stylo response to Mn toxicity were investigated using two stylo genotypes with contrasting Mn tolerance. Results showed that stylo genotype RY5 exhibited Mn tolerance superior to that of genotype TF2001, showing lower reductions in leaf chlorophyll concentration, maximum quantum yield of photosystem II (Fv/Fm) value and plant dry weight under Mn toxicity. Furthermore, RY5 tolerance to Mn toxicity could be attributed to stimulation of antioxidant protection and regulation of Mn homeostasis. Subsequently, a label-free quantitative proteomic analysis was conducted to investigate the protein profiles in the leaves and roots of stylo in response to Mn toxicity. A total of 356 differentially expressed proteins (DEPs) were identified, including 206 proteins from leaves and 150 proteins from roots, which consisted of 71 upregulated, 62 downregulated, 127 strongly induced and 96 completely suppressed proteins. These DEPs were mainly involved in defense response, photosynthesis, carbon fixation, metabolism, cell wall modulation and signaling. The qRT-PCR analysis verified that 10 out of 12 corresponding gene transcription patterns correlated with their encoding proteins after Mn exposure. Furthermore, stylo plants coped with Mn toxicity may be through enhancement of defense response and phenylpropanoid pathway, adjustment of metabolic process, and modulation of protein synthesis and turnover. Taken together, this study increases our understanding of tropical legume responses to Mn toxicity.