Mature Chinese mitten crabs, Eriocheir sinensis, are exposed to brackish water or coastal waters as an obligatory part during adult reproductive migration. In this study, we investigated the changes in serum total protein and oxyhemocyanin contents, alkaline phosphatase (AKP), superoxide dismutase (SOD) and phenoloxidase (PO) activities in both mature male and female E. when in freshwater and after step-wise acclimation to the following salinities: 7, 14, 21, 28, 35. Increasing salinities caused a gradual decrease in serum total protein and oxyhemocyanin contents. Serum AKP activity was elevated slightly at 7, thereafter it dropped slowly by the end of experiment. Serum SOD activity showed a slight decrease after increase of salinity until 21, it was activated above 21. Serum PO activity decreased gradually with the increase of salinity in males; however, it maintained a stable level above 21 in females. All tested parameters in serum except for SOD were significantly higher in females than those in males. It is suggested that elevated salinity induced a decrease in serum total protein and oxyhemocyanin contents. Serum immune enzyme activities in mature E. sinensis will be affected when the salinity was near to or above 28, which could exert a negative effect on reproduction. Further studies are needed to understand the biochemical adaptation of mature E. sinensis, during their reproductive migration acclimation to elevated salinities. Sex differences should not be ignored.