Abstract Pleurotus eryngii, also known as thistle mushroom, is rich in proteins, dietary fibre and polysaccharides. It has been developed as a functional food due to its antioxidant, anti-fatigue, anti-viral and anti-tumour functions. The objective of this study was to investigate the mechanisms of the immunoregulatory and antioxidant effects of P. eryngii superfine powder (PESP) in vivo. Test mice were fed with a diet containing 6.67% PESP to explore its effect on mouse delayed-type hypersensitivity, phagocytic index, serum hemolysin, serum and liver glutathione peroxidase (GSH-PX) activities, serum superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in liver homogenate. For the human study, volunteers received PESP at doses of 5, 10 or 15 g. Serum levels of 39 cytokines were determined before and after ingestion using a Millipore Human Kit and a liquid chip scanner. Our results showed that, in mice, dietary P. eryngii supplementation significantly increased the weight difference of ears (P < 0.01), phagocytic index (P < 0.05), serum hemolysin (P < 0.01), serum and liver GSH-PX activities (P < 0.01) and serum SOD activity (P < 0.01), but decreased MDA content (P < 0.01) in liver homogenate. In human trials, 5 g of PESP significantly decreased serum epidermal growth factor, while 15 g of PESP significantly decreased serum granulocyte–macrophage colony stimulating factor, macrophage-derived chemokine and tumour necrosis factor-α. No change was detected in serum cytokines after the administration of 10 g of PESP. P. eryngii can regulate innate and acquired immune functions in mice to execute antioxidant and anti-aging functions, and can alter human serum cytokines. The dual immunoregulatory effects of P. eryngii are consistent with the theory of traditional Chinese medicine.