Several groups of CD4⁺ T lymphocytes, known as Th1, Th2, Treg, and Th17, have currently been identified and well studied. Methods based on targeted regulation of differentiation of Th lymphocytes, which perform the immune response polarization, attract attention of scientists dealing with correction of immunemediated diseases. In the present study, endogenous β-galactoside-binding protein of the lectin family, galectin-3, was investigated as a regulator of T-cell homeostasis. Galectin-3 is known to be actively produced by tumor cells in malignant transformation and able to influence the processes of signal transduction, cell-cell cooperation, and the implementation of programmed cell death. As cell differentiation processes are directly connected with the regulation of gene expression, we investigated the effect of recombinant galectin-3 on expression of mRNA of transcription factors that guide differentiation of CD4⁺ lymphocytes. The study was performed using mononuclear cells from the peripheral blood of healthy individuals. The levels of gene expression were evaluated by real-time PCR. In the in vitro experiments, recombinant galectin-3 (0.5 mg/mL) up-regulated mRNA expression of transcription factors Gala-3 and Rorc and down-regulated mRNA expression of transcription factors T-bet and FoxP3. Up to a concentration of 1 mg/mL, recombinant galectin-3 stimulated Th-cell in a dose-dependent manner, whereas at higher concentrations, the stimulating effect weakened and inhibitory effect started prevailing. Thus, one can suppose that, through the regulation of lymphocyte differentiation, galectin-3 promotes the development of allergic, autoimmune, and neoplastic diseases, which allows us to consider galectin-3 as a potential target for therapy of these diseases.