Recent studies provide solid evidence for the importance to delineate the combined transplantation of neural stem cells (NSCs) and olfactory ensheathing cells (OECs) for the repair of central nervous system injury. One of the limitations of this approach is that the proportion of neurons differentiated from NSCs still remains at low level. Thus, how to induce NSCs to differentiate into neurons more efficiently by OECs is an attractive problem, which needs attention to be resolved. In the present study, we investigated the effects of hyperthermia-conditioned OEC-conditioned medium (OCM) on induction of NSCs into nerve cells. The conditioned medium, named 37OCM, 40OCM, and 40OCM+R (treated with HIF-1a inhibitor), were collected after OECs had been incubated at 37°C or 40°C for 6 hr, followed by incubation at 37°C for 42 hr; then, these conditioned medium were collected and used for NSC induction. Results of the present study demonstrated for the first time that 6 hr of hyperthermia (40°C) could induce OECs upregulation and the expression of hypoxia-inducible factor-1 alpha (HIF-1α). Moreover, compared with 37OCM and 40OCM+R, 40OCM could induce NSCs differentiation into neurons more efficiently, and this phenomenon is associated with the upregulation of HIF-1α after hyperthermia conditioning. The data implied that the secretory protein in OECs-cultured medium and upregulation of HIF-1α expression and binding activity induced by hyperthermia-conditioned OECs have synergistic effect to induce NSCs differentiation into a neural lineage.