BACKGROUND AND PURPOSE: The underlying transcriptomic signatures driving brain functional alterations in MS and neuromyelitis optica spectrum disorder (NMOSD) are still unclear. MATERIALS AND METHODS: Regional fractional amplitude of low-frequency fluctuation (fALFF) values were obtained and compared among 209 patients with MS, 90 patients with antiaquaporin-4 antibody (AQP4)+ NMOSD, 49 with AQP4- NMOSD, and 228 healthy controls from a discovery cohort. We used partial least squares (PLS) regression to identify the gene transcriptomic signatures associated with disease-related fALFF alterations. The biologic process and cell type-specific signature of the identified PLS genes were explored by enrichment analysis. The correlation between PLS genes and clinical variables was explored. A prospective independent cohort was used to validate the brain fALFF alterations and the repeatability of identified genes. RESULTS: MS, AQP4+ NMOSD, and AQP4- NMOSD showed decreased fALFF in cognition-related regions and deep gray matter, while NMOSD (both AQP4+ and AQP4-) additionally demonstrated lower fALFF in the visual region. The overlapping PLS1- genes (indicating that the genes were overexpressed as regional fALFF decreased) were enriched in response to regulation of the immune response in all diseases, and the PLS1- genes were specifically enriched in the epigenetics profile in MS, membrane disruption and cell adhesion in AQP4+ NMOSD, and leukocyte activation in AQP4- NMOSD. For the cell type transcriptional signature, microglia and astrocytes accounted for the decreased fALFF. The fALFF-associated PLS1- genes directly correlated with Expanded Disability Status Scale of MS and disease duration across disorders. CONCLUSIONS: We revealed the functional activity alterations and their underlying shared and specific gene transcriptional signatures in MS, AQP4+ NMOSD, and AQP4- NMOSD.