Abstract: To study the effect of the secondary metabolite Af-8 16-O-deacetylhelvolic acid 21, 16-lactone isolated from the endophytic Aspergillus fumigatus of the mangrove plant Rhizophora mucronata on the immunosuppressive activity in mouse spleen cells, CCK-8 cell viability determination method, calcineurin activity inhibition method in vitro, enzyme-linked immunosorbent assay（ELISA） and Western blot were used to study the effect of Af-8 on the NFAT signaling pathway in spleen cells. At concentrations resulting in relatively low cytotoxicity(IC₅₀ =（168.96±1.35） μmol/L) Af-8 significantly inhibited calcineurin activity with its inhibition rate reaches 88.81% and ConA-induced splenocytes proliferation in a dose-dependent manner with IC₅₀ value of（12.11±0.11） μmol/L. The secretion of interleukin-2（IL-2） were down-regulated by Af-8 in cell supernatant as determined by ELISA. Western blotting revealed that Af-8 had a significant inhibitory effect on the dephosphorylation of NFAT1 protein in mouse T lymphocyte. The results showed that Af-8 affects the NFAT signaling pathway by inhibiting CN enzyme, thereby exerting an immunosuppressive effect.