Glioblastoma (GBM) is a typical malignant tumor, and there are no effective drugs capable of improving patient survival. Docosahexaenoic acid (DHA), a nutrient essential to animal health and neurodevelopment, exerts an anticancer effect in several types of cancer. However, the function of DHA in GBM is still unclear. Here, we showed that DHA could repress the migration and invasion of GBM U251 cells and promote their apoptosis in a dose- and time-dependent manner, indicating that DHA has an anticancer effect on GBM cells. Whole-transcriptome analysis indicated that DHA treatment mainly regulates the genes associated with receptor binding, oxidoreductase activity, organic acid transmembrane transporter activity, and carboxylic acid transmembrane transporter activity. Long non-coding RNAs (LncRNAs) involved in the regulation network of DHA were also identified, and their targets were assigned to the Gene Ontology (GO) categories. In silico analysis was conducted to predict the pathways related to the differentially expressed genes by DHA treatment. Our findings suggest that DHA acts as an antitumor agent in GBM, which may provide a suitable means of improving the efficacy of GBM treatment in the future.