Background: Although reports suggest Chinese herbal medicine treatment of ovarian cancer (OC) has a good effect, the role of isorhamnetin (ISO), a flavonol aglycone with immune, anti-inflammatory, cardiovascular and cerebrovascular protective effects, as well as an anticancer effect, in OC remains unclear.Network pharmacology was used to explore this in vitro and in vivo, and to identify relevant targets. Methods:The common targets of ISO in the treatment of OC were screened by constructing drug targets and disease gene databases for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The protein-protein interaction network was constructed by STRING.Overlapping targets were further analyzed using the online tool UALCAN to analyze the correlation between gene expression and patient survival and prognosis. The effect of ISO on OC cell proliferation, migration, and invasion was assessed in vivo and in vitro, and the function of the estrogen receptor 1 (ESR1) in the development of OC was examined by overexpressing and knocking down ESR1 expression.Results: Through network pharmacology analysis, 25 target genes related to ISO-OC were screened out.The overall survival rate of OC patients only significantly correlated with high expression of ESR1 among 13 highly expressed overlapping genes. ISO significantly inhibited the proliferation, migration and invasion of OC cells in vitro and inhibited tumor growth in vivo. Overexpression of ESR1 significantly promoted the proliferation, migration and invasion of OC cells, whereas knockdown of ESR1 showed the opposite result.In addition, overexpression of ESR1 significantly reversed the inhibitory effect of ISO on the proliferation, migration and invasion of OC cells. Conclusions:We confirmed that ISO inhibits OC cell proliferation, migration and invasion by targeting ESR1 expression, which provides a theoretical basis for further pharmacological research.