Objective To explore the expression and prognostic significance of histone demethylase JMJD2B and HIF-1α in rhabdomyosarcoma(RMS) tissues. Methods A total of 78 RMS patients were followed up to obtain survival time, and immunohistochemical staining was used to detect the expression of JMJD2B and HIF-1α in RMS tissues and analyze their relation with clinicopathological characteristics and prognosis. Results Among the 78 samples, there were 46 (59.0%) and 44 (56.4%) cases of positive expression of JMJD2B and HIF-1α, respectively. The expression of JMJD2B and HIF-1α were positively correlated (P < 0.001, r=0.518) and related to tumor risk, metastasis, IRS staging and neoadjuvant chemotherapy (all P < 0.05). The Kaplan-Meier survival curve and univariate Cox regression analysis showed that the risk level, neoadjuvant chemotherapy, the expression of HIF-1α and JMJD2B were related to the prognosis of patients (all P < 0.05). Multivariate Cox regression analysis showed that JMJD2B (HR=3.161, P=0.039) and risk degree (HR=2.925, P=0.001) could be used as independent prognostic factors for RMS patients. Conclusion JMJD2B and HIF-1α expression are both significantly related to the overall survival of rhabdomyosarcoma patients, and JMJD2B is an independent prognostic indicator.