Agaricus bisporus (A. bisporus) has fragile tissues and is highly susceptible to post-harvest decay and spoilage, which affecting the development of the industry. Ultraviolet B (UV-B) irradiation, as a typical irradiation preservation technology, is effective in inducing the production of endogenous metabolic substances in organisms and enhancing their level of resistance. The objective of this study was to investigate the mechanism of activation of the antioxidant defence system in A. bisporus by UV-B irradiation, utilising a range of UV-B irradiation doses (0, 25, 50 and 100 kJ m−2). In this study we found that 50 kJ m−2 UV-B irradiation effectively delayed the accumulation of reactive oxygen species (ROS), inhibited NADPH oxidase (NOX) activity and the expression of Rbohf, PXMP2, PXMP4, APO, and MPV17. Moreover, it could increase the accumulation of ascorbate (AsA) and glutathione (GSH), enhance the activities of ascorbate peroxidase (APX) and glutathione peroxidase (GSH-PX), and it also effectively induce catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities and up-regulate the expression levels of related genes. In addition, we found that UV-B irradiation upregulated the expression of UVR8 and suppressed the expression of PEX5, PEX11 and PMD1. These results suggest that 50 kJ m−2 UV-B irradiation could stimulate the UV Resistance Loucs 8 (UVR8) receptor, regulate peroxisome proliferation, and enhance the ability of A. bisporus to resist oxidative stress, thereby maintaining the cellular redox homeostasis, this provides a new strategy for the study of extended postharvest storage stability of A. bisporus.