Solanum nigrum L. is one of the major medicinal plants used to treat cancer. However, the functional mechanism of S. nigrum L. extract is still unknown in spite of numerous studies on its active components. In this study, we probed the potential anticancer mechanism of the aqueous extract of S. nigrum L. (AESN) towards human breast cancer cell line MCF7. At a concentration of 10 g/L, AESN caused 43% cytotoxicity, inhibited the migration, and suppressed the activities of hexokinase and pyruvate kinase by about 30% and 40%, respectively, towards the MCF7 cells. RT²-PCR analysis of a panel of 89 caner-related genes identified 13 upregulated and eight downregulated genes (>2-folds) in MCF7 cells upon AESN treatment. Gene ontology (GO) and functional disease ontology (FunDO) analyses show that the antitumor function of S. nigrum L. involves multiple genes and these genes are shared across other diseases or disorders.