Radiation-induced brain edema is a serious adverse effect of radiotherapy. Although there are many causes of radiation-induced brain edema, the pathogenesis is not clear and clinical treatment is not ideal. Therefore, knowing the differential expression of the brain microvascular endothelial cell (BMEC) transcriptome after brain radiotherapy may shed light on the pathogenesis of radiation-induced brain edema. The present study used RNA-Seq technique to identify 383 BMEC transcripts differentially expressed (many 2-fold or higher; P < 0.05) between control and X-ray-treated primary cultured rat BMECs. Compared with controls, X-ray-treated BMECs had 183 significantly up-regulated transcripts and 200 significantly down-regulated transcripts. The differentially expressed genes were associated with the biological processes of the cell cycle, apoptosis, vascular permeability, and extracellular junctions. The functional changes identified in the X-ray-treated BMECs included Ca2+ signaling, phosphoinositide 3-kinase-Akt signaling, and methionine degradation. These results indicated that transcript expression was substantially affected by radiation exposure and the proteins encoded by these differentially expressed genes may play a significant role in radiotherapy-induced brain edema. Our findings provide additional insight into the molecular mechanisms of radiation-induced brain edema and may be helpful in the development of clinical treatment of this adverse reaction to radiotherapy. © 2020 The Author(s).