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Objective To explore the therapeutic mechanism of cordycepin for acute kidney injury （AKI） based upon gene chips in gene expression omnibus （GEO） database, network pharmacology, molecular docking and in vitro experiments. Methods Targeted genes of AKI were obtained from two databases, GEO （GSE87025） and GeneCards. Cordycepin-related targets were retrieved from three databases of TargetNet, BATMAN and GeneCards. The common targets of AKI and cordycepin were screened by taking the intersection of two sets. Molecular docking was performed for cordycepin and key targets after gene ontology （GO） annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. Cell experiments were performed for control, hypoxia/reoxygenation and hypoxia/reoxygenation+cordycepin groups for experimental verifications of the relevant targets. Results There were 12 common targets of AKI and cordycepin. GO functional annotation results indicated that these targets participated predominantly in negative regulation of apoptotic signaling pathway and necroptosis process. KEGG enrichment analysis results showed that they played some roles in signaling pathways of Toll-like receptor, necroptosis, nucleotide-binding oligomerization domain （NOD）-like receptor, nuclear factor-κB （NF-kB）, tumor necrosis factor （TNF） and cell apoptosis, etc. Optimal binding energy between cordycepin and receptor interacting protein kinase 1 （RIPK1） in molecular docking was -7.1, denoting potent binding activity. Western blot indicated that cordycepin down-regulated the expressions of RIPK1, bcl-2 associated x protein （Bax） and caspase 3 and up-regulated B cell lymphoma/lewkmia-2（Bcl-2）. Cell immunofluorescence results revealed that cordycepin down-regulated the expression of RIPK1. Conclusion Cordycepin may alleviate hypoxia/reoxygenation-induced renal tubular epithelial cell injury through regulating RIPK1-mediated apoptosis. Thus it achieves the therapeutic efficacy for AKI.