Objective：To construct rat p300 short hairpin RNA （shRNA） eukaryotic expression vector，and investigate the effect of silencing p300 gene on the apoptosis and activating transcription factor 3（ATF3） acetylation in rat glomerular messangial cells （GMCs） stimulated by sublytic C5b-9. Methods：Three kinds of shRNAs targeting p300 gene were synthesized and cloned into eukaryotic expression vector pGCsi-U6/Neo/GFP/shRNA. The recombinant plasmids were transfected into cultured GMCs by NeonTM transfection system. P300 protein in the transfected cells was detected by Western blot to find out the optimal shRNA against p300 gene. The cell apoptosis was measured by flow cytometry and the acetylation of ATF3 was evaluated by Western blot combined with immunoprecipitation （IP） assays. Results：It was verified by nucleotide sequencing that the constructed p300 shRNAs were correct. Western blot assay showed that the p300 shRNA-2 was able to silence the target gene most effectively. Knockdown of p300 by shRNA in the GMCs reduced the number of GMC apoptosis as well as the level of ATF3 acetylation induced by sublytic C5b-9. Conclusion：The rat eukaryotic expression vector p300 shRNA was successfully constructed. It was preliminarily confirmed that p300 could promote GMC apoptosis triggered by sublytic C5b-9 through p300-regulated ATF3 acetylation.