Objective: Rituximab (RTX) is a monoclonal antibody directed against CD20 epitope expressed on B cells used widely in neuromyelitis optica spectrum disorder (NMOsd). There are few studies evaluating anti‐aquaporin‐4 (AQP4) positive‐to‐negative seroconversion. In this series, we evaluated this phenomenon in a Latin American population. Methods: A retrospective, longitudinal and analytic study was carried out in 19 AQP4 immunoglobulin G (IgG)+ NMOsd patients. We monitored all patients with a yearly test for AQP4‐IgG serum autoantibodies. Potential relationships between negative seroconversion and annualized relapse rate, Expanded Disability Status Scale score, immunosuppressive or immunoregulatory treatment were assessed. Results: We included 19 patients, 17 (89.5%) were women. The mean Expanded Disability Status Scale score and annualized relapse rate at clinical onset was 3.8 (±1.97) and 0.81 (±0.577), respectively. Disease‐modifying treatment included RTX, cyclophosphamide, azathioprine and methotrexate. We documented positive‐to‐negative seroconversion in six of the patients in the RTX group (P = 0.047). No significant changes were observed in the annualized relapse rate (2.11 vs 1.88, P = 0.06) or Expanded Disability Status Scale scores (4.41 vs 3.1, P = 0.25) between the group of seroconverted patients from those who remained positive. Conclusions: Treatment with RTX in patients with seropositive AQP4‐IgG NMOsd can lead to negative conversion, but our findings do not support a clinical reflection from this change. The role of monitoring for AQP4‐IgG is yet to be defined in the Mexican NMOsd population. [ABSTRACT FROM AUTHOR]