At the moment it is unknown to what extent the impaired function of T lymphocytes in ESRD patients depends on uremia, and to what extent on hemodialysis (HD) procedure. Therefore, the purpose of the study was to evaluate percentages of T lymphocyte subpopulations ex vivo, plasma concentrations of IL12p70, TNF, IL-10, IL-6, IL-1β, IL-8 cytokines and selected proliferation parameters of in vitro activated T lymphocytes in HD patients before and after single HD procedure using flow cytometry. We demonstrated that the percentage of CD8+ cells ex vivo was decreased while the CD4+/CD8+ ratio was increased after HD procedure. Also, there was significant decrease in the percentage of CD8+HLA-DR+, CD8+CD69+ and CD8+CD95+ cells after HD. At the same time, an increase in the percentage of CD4+CD95+ cells was observed after HD. From all analyzed cytokines, only the concentration of IL-8 was significantly decreased after HD procedure. A single HD session enhanced proliferation capacity of CD4+ cells but not CD8+ cells in vitro by increasing number of cell divisions and percentage of dividing cells. Our results show that a single hemodialysis can have immunomodulatory effect on HD patients and may contribute to the state of immune deficiency observed in patients with ESRD.