Background: There is a close relationship between neutrophil extracellular traps (NETs) and venous thromboembolism (VTE). The regulatory role and mechanism of glucocorticoids (GC) in the formation of NETs are unclear. Objective: This study was conducted to assess the effect of GC on the formation of NETs. Methods: We constructed a mouse VTE model and treated them with GC to observe the effect of GC on the formation of NETs. In this regard, peripheral blood neutrophils were isolated, and the effect and mechanism of GC in neutrophil activation were analyzed. Results: Following LPS treatment, the colony-forming ability of neutrophils and their ability to form NETs increased significantly. The analysis of cytokine changes by RT-PCR combined with ELISA showed that the level of inflammatory factors in LPS-activated neutrophils increased significantly; however, these factors were significantly inhibited after GC treatment, and the inhibitory effect was positively correlated with the concentration of GC. LPS treatment was able to activate the production of ROS and lipid peroxides, however, this activation was significantly inhibited after GC treatment, and the inhibition increased with increasing doses of GC. Further examination of the changes in NF-κB signaling activation revealed that LPS-induced NF-κB signaling was significantly inhibited after GC treatment, and this inhibition increased with increasing the GC concentration. Conclusion: Glucocorticoids were able to inhibit neutrophil activation and reduce the formation of NETs. The research results provided a new research direction for clinical antithrombotic treatment.