Objective To evaluate the influence of Fasudil on the lipopolysaccharide (LPS)-induced activation and inflammatory response of astrocytes and TLR4/ NF-κB signaling pathway. Methods Astrocytes were separated from the cerebral cortex of newly-born C57BL/6 mice and cultured in vitro. The cells were divided into PBS control group, 1 μg/mL LPS stimulation group, 1 μg/mL LPS combined with 15 μg/mL Fasudil treatment group. The production of NO in the cell supernatant was detected by Griess reagent. The levels of tumor necrosis factor-α (TNF-α), interleukin -6(IL-6), IL-10 and IL-4 were measured by ELISA. The expressions of glial fibrillary acidic protein (GFAP) and TLR4 in the astrocytes were detected by immunofluorescence cytochemistry. The protein levels of GFAP, TLR4 and p-NF-κBp65 were assessed by Western blot analysis. Results Compared with the PBS control group, the levels of NO, TNF-α and IL-6 in the LPS group significantly increased, but the levels of IL-10 and IL-4 decreased. Fasudil treatment significantly inhibited LPS-induced the secretion of NO, TNF-α and IL-6 and enhanced the production of IL-10 and IL-4. The expression of GFAP significantly decreased in the Fasudil treatment group, and the protein levels of TLR4 and p-NF-κBp65 also decreased. Conclusion Fasudil can inhibit LPS-induced astrocyte activation and inflammatory response by blocking TLR4/NF-κB signaling pathway.