Intravenous methylprednisolone (IVMP) is the gold standard treatment in acute relapses of multiple sclerosis. Knowing the response to IVMP in advance could facilitate earlier selection of patients for subsequent courses of therapy. However, molecular mechanisms and changes in gene expression induced by methylprednisolone remain unknown. The aim of the study was to identify in vivo differentially expressed genes in relapsing-remitting multiple sclerosis patients after 3-6 days of treatment with IVMP. For this purpose, whole-genome transcription profiling of CD4+ T lymphocytes was performed before and after treatment with IVMP in 8 relapsing-remitting multiple sclerosis patients during relapse using Human GE 4x44K v2 microarrays. Differentially expressed genes were identified using a paired t test on GeneSpring v13.0 software. A P-value < 0.001 and a twofold change were considered significant. Microarray data were confirmed using real-time PCR. Microarray revealed changes in gene expression: four genes were downregulated (B3GNT3, ZNF683, IFNG and TNF) and seven upregulated (DEFA4, CTSG, DEFA8P, AZU1, MPO, ELANE and PRTN3). Pathway analysis revealed the transforming growth factor-β signaling pathway to be affected. Comparison with previously published data on in vitro methylprednisolone-regulated genes showed that SMAD7, TNF and CHI3L1 were also downregulated in vivo in relapsing-remitting multiple sclerosis patients. In summary, we performed the first in vivo transcriptome analysis in CD4+ T lymphocytes before and after the treatment with IVMP in patients with multiple sclerosis. Identification of differentially expressed genes in patients receiving IVMP could improve our understanding of the molecular mechanisms underlying the therapeutic effects of IVMP and highlight potential biomarkers of the response to IVMP.