javascript:void(0) Antioxidants are known for their health beneficial effects by reducing oxidative damage, increasing free radical scavenging rate, blocking and terminating oxidation reactions. They have been widely used as supplements in food, cosmetics, nutraceuticals and medicines. Emerging researches increasingly indicate that the development of diverse research methodologies and the exploration of new microorganisms for heterogeneous synthesis of antioxidants play an indispensable role in socioeconomic development. In this study, we engineered Saccharomyces boulardii for probiotic supplementation of antioxidants such as L-ergothioneine (EGT). We first constructed double knockout of ura3 and trp1 gene in S. boulardii, to facilitate plasmid-based expressions. To further enable effective genome editing of S. boulardii, we implemented the PiggyBac system to transpose the heterologous gene expression cassettes into the chromosomes of S. boulardii. By using enhanced green fluorescent protein (EGFP) as the reporter gene, we achieved random chromosomal integration of EGFP expression cassette. EGT-producing strains were subsequently obtained via the PiggyBac transposon system. One isolated S. boulardii mutant produced EGT at 17.50 mg/L after 120 h cultivation. In summary, we have applied the PiggyBac transposon system to S. boulardii for the first time for genetic engineering. The engineered probiotic yeast S. boulardii has been endowed with new antioxidant properties and produces EGT. It has potential applications in developing novel therapeutics and dietary supplements for the prevention and treatment of gastrointestinal disorders.