Objective: To investigate the cytotoxic effect of RSK inhibitor BI-D1870 in N2a and determine the concentration and time of cytotoxic effect. Methods: N2a cells were cultured in vitro, and the specific proteins NeuroD1, NeuN and DCX were identified by immunofluorescence. N2a cells were exposed to a series concentration of 10 μmol•L-1 to 80 μmol•L-1 BI-D1870 and a series duration of 4 h to 24 h. The morphology of N2a cell was observed with the inverted microscope, and the cell viability was measured with CCK-8 method, and lactate dehydrogenase (LDH) release of cell culture medium was assayed by pyruvate reduction method. Results: NeuroD1, NeuN and DCX were detected in N2a cells by immunofluorescence. Compared with the DMSO solvent control group, with the increase of BI-D1870 concentration and the exposure prolongation of BI-D1870, the damage degree of N2a cells gradually increased. The cell morphology changed abnormally, the cell viability decreased gradually (P<0.05) and LDH level in culture medium went up and then went down (P<0.05). Under the treatment of 40 μmol•L-1 for 12 h, the swelling of N2a cells was obvious, the refraction was reduced, the cell viability was about 60%, and the LDH release in culture medium was the highest. Conclusion: N2a cells have the characteristics of neuron. BI-D1870 has cytotoxic effect for N2a cells and the optimal cytotoxic condition is 40 μmol•L-1 for 12 h.