Administration of ozone (O3) is often used in the treatment of low back pain. Administration of O3 can, however, cause neurotoxicity in spinal cord neurons via induction of endoplasmic reticulum (ER) calcium (Ca2+) release and activation of the Ca2+/calmodulin‑dependent protein kinase II (CaMKII)/mitogen‑activated protein kinase (MAPK) pathway. The aim of the current study was to confirm whether administration of O3 causes ER stress and if the consequential overexpression of adenovirus‑mediated spliced X box binding protein 1 (XBP1s), which is the effector of ER stress and a crucial transcriptional factor gene in charge of cell survival, has a protective effect on spinal cord neurons after O3 exposure. To address this aim, the expression of GRP78, an ER chaperone and signaling regulator, and the expression of XBP1s in rat primary spinal cord neurons that underwent O3 exposure were investigated. Primary neurons exposed to O3 exhibited increased GRP78 and XBP1s expression levels. Interestingly, the effect of decreased neuron viability was blocked when cells were pretreated with Adv‑XBP1s. Moreover, overexpression of XBP1s suppressed cell death caused by O3 exposure. These results suggest that overexpression of activated XBP1s protects against neuronal cell death following O3 exposure and that activation of the XBP1s pathway may offer a preventative way for prophylactic treatment of spinal cord neurons exposed to O3.