Hepatitis B virus (HBV) infection is one of the major causes of chronic liver inflammation. Tim-3 acts as a negative regulatory molecule and plays a critical role in immune tolerance. In the current study, we investigated Tim-3 expression on peripheral monocytes and CD3+CD16/CD56+ natural killer like T (NKT-like) cells in chronic hepatitis B (CHB) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from 52 CHB patients and 60 healthy controls. Tim-3+CD14+ cells and Tim-3+CD3+CD16/CD56+ cells were analyzed by flow cytometry. Results showed that expression of Tim-3 was significantly increased on both the monocytes and NKT-like cells in CHB patients than in controls (P = 0.002 and P < 0.001, respectively). Tim-3 levels on monocytes and NKT-like cells were further upregulated in patients with acute-on-chronic liver failure (ACLF). In addition, we assessed the correlation of Tim-3 expression with levels of alanine aminotransferase (ALT) and tumor necrosis factor alpha (TNF-α). Data revealed that Tim-3 expression on both monocytes and NKT-like cells was positively correlated with level of ALT (r = 0.59, P < 0.001, and r = 0.60, P < 0.001, respectively), whereas Tim-3 expression on NKT-like cells was negatively correlated with serum level of TNF-α (r = −0.54, P < 0.001) in CHB patients. Our results suggest that Tim-3 may play important roles in the pathogenesis of CHB.