Abstract: Objective To investigate the effect of lentiviral vector-mediated Runx3 overexpression on Th cell differentiation in patients with chronic hepatitis B (CHB). Methods Peripheral CD4 + T cells derived from 29 CHB patients were transfected with the recombinant lentiviral vector (pGC-FU-Runx3) and the negative control lentiviral vector (pGC-FU) , respectively. Then the cell culture supernatants and the CD4 + T cells were collected on the 5th day. The expression of Th1-type cytokine (IFN-γ) and Th2-type cytokine (IL-4) were measured by ELISA and the expression of T-bet and GATA3 mRNA were assayed by quantitative real-time PCR. Results ①Compared with the pGC-FU transfected group, the expression of IFN-γ in the pGC-FU-Runx3 transfected group significantly increased (P = 0.003). ②Compared with the pGC-FU transfected group, the expression of IL-4 in the pGC-FU-Runx3 transfected group significantly decreased (P = 0.007). ③Compared with the pGC-FU transfected group, the ratio of IFN-γ/IL-4 in the pGC-FU-Runx3 transfected group significantly increased (P = 0.001). ④There was no difference in T-bet and GATA3 mRNA expression between the pGC-FU transfected group and the pGC-FU-Runx3 transfected group (both P ＞ 0.05). However, compared with the pGC-FU transfected group, the ratio of T-bet/GATA3 in the pGC-FU-Runx3 transfected group significantly increased (P = 0.005). Conclusions Runx3 overexpression can promote the secretion of Th1-type cytokines and inhibit the secretion of Th2-type cytokines in CHB patients. It can also induce Th cell differentiating into Th1 cell lineage and improve the Th1/Th2 imbalance in CHB patients.