Unexplained infertility (UI) is one of the most common diagnoses in the fertility care. Seminal plasma (SP) plays a crucial role in the regulation of female immune responses and the success of a pregnancy. In vitro fertilization (IVF) is a well-known method for the treatment of UI. In this study, we aimed to investigate the effect of SP on the differentiation of T helper (Th) cell subsets and the relationship between these subsets with the rate of IVF success in a group of women complicated with UI compared to those with normal pregnancy. This study was conducted on 20 UI couples (ten with successful and ten with unsuccessful IVF outcome) and 10 fertile couples as the control group. Four color flow cytometry technique was used to detect Th cell subsets in the peripheral blood mononuclear cells (PBMC) with or without stimulation by SP. Results indicated that the frequencies of IL-17+ and Foxp3+ T cells after incubation with SP was significantly increased in couples with unsuccessful IVF outcome as compared to successful and healthy groups (p<0.05). Additionally, a positive correlation was observed between Th1 and Th2 cells in the unsuccessful IVF group (R=0.6, p=0.03). In summary, the results of the present study demonstrated that exposure to SP might increase Th17 and Treg cell frequencies in infertile women with unsuccessful IVF, and might also balance inflammatory to regulatory responses to finally tune-up the Th1/Th2/Th17/Treg balance and support the success of IVF.