The population size of horseshoe crab is decreasing year by year in China. In order to achieve the protection and sustainable utilization of horseshoe crab resources, it is urgent to study the hematopoietic mechanism of the horseshoe crab, and to make the rapid recovery of hemopoietic lymphocytes from the horseshoe crab. Therefore, strengthening the recovery of horseshoe crabs blood lymphocytes is expected to reduce the loss of horseshoe crabs wild resources. In this study, N-acetylcysteine (NAC) and Vibrio anguillarum (V) were injected into horseshoe crab to compare the changes of reactive oxygen content and non-specific immuno enzyme activity in total lymphocytes of Chinese horseshoe crab at 0, 6, 12, 24, 48 hours after injection. The results showed that compared with the control group, the total hemocyte counts (THC), reactive oxygen species (ROS), malonyldialdehyde (MDA) of horseshoe crabs after NAC injection showed downward trends, while the activity of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), lysozyme (LZM), and alkaline phosphatase (AKP) showed upward trends. Under the stimulation of NAC and V. anguillarum at different concentrations, the decrease trend of THC, ROS, MDA in horseshoe crab was slowed down compared with single injection of NAC, while the activities of other enzymes were increased. However, HC showed no significant change during the whole experiment. At 6-48 hours, THC, ROS in the NAC group showed a decreasing trend compared with the V. anguillarum group and Co-stimulation groups between NAC and V.anguillarum, the SOD activity of NAC and V. anguillarum was significantly higher than other groups. At 48 hours, CAT, T-AOC showed no significant difference at the NAC group, V. anguillarum group and co-stimulation groups, but both of them were significantly higher than the control group. The NAC+10⁶V group had the lowest MDA in 48 hours. The activity of AKP increased from 12 to 48 hours, and the activity of LZM in NAC group was the highest at 48 hours. There is a negative correlation between THC, ROS, MDA, HC and SOD, CAT, T-AOC, LZM, AKP. NAC can lower the value of horseshoe crab THC, ROS, NAC and co-stimulation with the V. anguillarum can mitigate the decline. However, the above results showed that inactivated V. anguillarum can increase THC, ROS of horseshoe crab，and antioxidant and immunoenzyme activities were increased in all treatment groups. Therefore, it can be concluded that NAC can reduce THC, ROS of horseshoe crab and co-stimulation of N-acetylcysteine with V. anguillarum can alleviate the decreasing trend，however, inactivated V. anguillarum could increase THC, ROS of horseshoe crab, and the activity of antioxidant and immunoenzyme was increased in all treatment groups. This indicated that when NAC reduced ROS content in horseshoe crab, hemolytic cells in the body also decreased, while NAC combined with V. anguillarum inactivated, ROS, THC decreased less than the NAC group. It can be concluded that NAC and V. anguillarum can stimulate the immune functions of horseshoe crabs, meanwhile, ROS may play an important role in the proliferation and regeneration of blood lymphocytes of the horseshoe crabs.