OBJECTIVE Monotherapy with pegylated interferon-α (Peg-IFNα) or the nucleos(t)ide analogs (NA) currently approved for treating chronic hepatitis B (CHB) has limited efficacy. Studies on the combination of Peg-IFNα/NA have shown conflicting results. We investigated whether sequentially adding on Peg-IFNα to tenofovir enhances serological response rates. METHODS Treatment-naïve, hepatitis B envelope antigen (HBeAg)-positive CHB patients with moderately elevated alanine aminotransferase (ALT; 48-200 IU/mL) were started on tenofovir (300 mg/day) and enrolled at week 12 in a 1:1 ratio to either receive Peg-IFNα2b add-on (1.5 μg/kg/week) from week 12 to 36 (n = 53) or continue tenofovir monotherapy (n = 53). Both treatment arms received tenofovir consolidation therapy until week 72. The primary end-point was HBeAg loss at week 72. RESULTS At week 72, the rate of HBeAg loss was higher in the Peg-IFNα2b add-on group (35.8%) compared to the tenofovir monotherapy group (17%) (P = 0.028; odds ratio, 2.73, 95% confidence interval, 1.09-6.79), and considerably higher in patients with a baseline hepatitis B virus (HBV)-DNA level >6 log IU/mL (32.6% vs 11.4%; P = 0.021). Rates of HBV-DNA loss (77.4% vs 71.7%; P = 0.51), ALT normalization (62.3% vs 52.8%; P = 0.32), and sustained virologic response (20.8% vs 11.3%; P = 0.18) at week 72 were comparable between the two groups. Significantly more patients in the add-on group had >3 log HBV-DNA reduction at week 36 (92.5% vs 66%; P = 0.001). Four patients treated with Peg-IFNα2b add-on achieved hepatitis B surface antigen (HBsAg) loss compared with one patient receiving tenofovir monotherapy. Decline of HBV-DNA of >2 log at week 4 led to higher HBeAg loss at week 72, independent of treatment arm. No patient had treatment-related adverse effects requiring treatment discontinuation. CONCLUSIONS Twenty-four weeks of Peg-IFNα2b as an add-on sequential regimen to tenofovir is safe and resulted in greater loss of HBeAg and HBsAg compared to tenofovir monotherapy in selected HBeAg-positive patients. Viral load reduction followed by immune modulation is a potentially useful approach.