Ethnopharmacological relevance Cordyceps sinensis, an entomogenous fungus used in traditional Chinese medicine with multiple pharmacological activities. However, its usage has been limited due to the high price and short supply. Isolate of fungi strains from natural Cordyceps sinensis to achieve a large-scale production by fermentation is an alternative choice. The aim of this study was to investigate and compare the effects of mycelia extracts of different fungal stains isolated from natural Cordyceps sinensis on macrophage functions in vitro. Materials and methods Macrophages' proliferation, phagocytosis, nitric oxide (NO) production, cytokines secretion, iNOS, NF-κB p65 activation and translocation were investigated by the MTT assay, flow cytometry assay, Griess reagent method, ELISA, western blot and immunostaining assay, respectively. Results The results showed that the effects of cultured Cordyceps mycelia of different fungal strains isolated from natural Cordyceps sinensis on macrophages greatly variant. Among 17 Cordyceps aqueous extracts, only five extracts (UM01, QH11, BNQM, GNCC and DCXC) could significantly increase cell proliferation and NO production of RAW 264.7 mouse macrophages. Moreover, the five extracts, especially UM01 and QH11, significantly enhanced phagocytosis and promoted cytokines release of macrophages. Polysaccharides in cultured UM01 mycelia were found to be the main immune stimulating compounds. Conclusions The variation of biological effects of fermented mycelia of different fungal strains from natural Cordyceps sinensis may be derived from their chemical diversity, especially polysaccharides, which need further study in future. © 2013 Elsevier Ireland Ltd.