Background: Rituximab has been used successfully in the recent years for treatment of neuromyelitis optica spectrum disorders (NMOSD). However, a uniform treatment protocol for maintenance therapy and the best interval for evaluation and retreatment have not been postulated. We evaluated the efficacy and safety of rituximab treatment as second line therapy, in Iranian patients with refractory NMOSD, based on annualized relapse rate (ARR) and expanded disability status scale (EDSS). Methods: In this prospective before-after study, a total of 18 patients were treated with a loading dose of rituximab (375 mg/m2 weekly in 4 consecutive weeks). Flow cytometric determination of CD19+ B cell in peripheral blood sample was carried every 6 weeks and patients were re-treated based on B cell repopulation with a single dose of 375 mg/m2. Wilcoxon signed rank test was used to evaluate the ARR and EDSS before and after treatment. A p-value of <0.05 was considered statistically significant. Results: Of the 18 patients, 10 (55.5%) were relapse-free during the period of follow up. The EDSS scores were reduced in nine (50%) patients and stable in the remaining nine (50%). The mean EDSS score before and after treatment were 4.1±0.4 and 3.7±0.3, respectively, which was statistically significant. There was also a statistically significant reduction in median ARR after treatment (1.48 (range 0.47-5) vs. 0 (range 0-2)). Rituximab administration did not have significant adverse effect in 94% of patients. Conclusion: Repeated treatment with Rituximab is an effective and well-tolerated treatment in refractory NMOSD.