Regulatory T lymphocytes (Tregs) play an important role in host immune homeostasis. During PRRSV infection, increased number of Tregs was linked to delayed and weak antiviral immunity. However, whether PRRSV could induce Treg proliferation in the naïve lymphocyte subpopulation remains controversial. This study aimed to determine whether PRRSV could induce Treg proliferation in porcine naïve leukocyte subpopulation. BrDU labeling system was utilized for determination of cellular proliferation in the culture system. PBMCs isolated from PRRSV-negative crossbred pigs were cultured with 0.1 m.o.i. of PRRSV, or mock-infected MARC-145 cell lysate for 96 hours. The cells were further subjected to immunofluorescent stainings. The presence of PRRSV significantly enhanced proliferation of CD4+CD25high subpopulation, compared to the control. The majority of detected Tregs (CD4+CD25highFOXP3+) exhibited proliferative marker (BrDU+). In this study, identification of porcine Tregs was confirmed using 2 anti-FOXP3 mAbs, including anti-mouse/rat FOXP3 (clone FJK-16s, IgG1) and anti-human FOXP3 (clone 236A/E7, IgG1). Comparable percentages of porcine FOXP3+ cells could be detected by the two mAbs. In another experiment, peripheral blood lymphocytes (PBLs) from PRRSV-negative crossbred pigs were co-cultured with autologous PRRSV-pulsed monocyte-derived dendritic cells (MoDCs) for 96 hours. Subsequently, the cells were subjected to immunofluorescent staining. Consistent to the findings in the PBMCs, PRRSV-pulsed MoDCs could induce proliferation of the CD4+CD25high and Treg (CD4+CD25highFOXP3+) subpopulations in the culture system. Our findings indicated that PRRSV could induce Treg proliferation in the naïve porcine lymphocyte subpopulation. The results also confirmed that both anti-mouse/rat and anti-human FOXP3 mAbs could be used for identifying porcine Tregs. The findings of PRRSV induced Treg proliferation in the naïve leukocyte subpopulation explained an increased numbers of Tregs during PRRSV infection and provided better understanding in PRRSV immunopathology.