Background & Aims: Inconsistencies in results and guideline recommendations regarding the durability of nucleos(t)ide analogue-induced hepatitis B e antigen (HBeAg) seroconversion require clarification. We studied the long-term durability of nucleos(t)ide analogue-induced HBeAg seroconversion in patients with chronic hepatitis B virus (HBV) infection. Methods: We performed a single-center cohort study of 132 HBeAg-positive patients who had received nucleos(t)ide analogue therapy. Results: During a median treatment duration of 26 months (range, 16-43 mo), HBeAg seroconversion occurred in 46 of 132 subjects (35%). Forty-two subjects (91%) had follow-up evaluation after HBeAg seroconversion. During a median follow-up period of 59 months (range, 28-103 mo) after HBeAg seroconversion, 13 of 42 patients (31%) showed a durable remission (defined as HBeAg negative and HBV-DNA level <10,000 copies/mL). Overall, 33 of 42 subjects (79%) continued therapy after HBeAg seroconversion, of these, 22 (67%) showed serologic and/or virologic recurrence. Nine of 42 subjects (21%) discontinued therapy after HBeAg seroconversion and at least 6 months of consolidation therapy. Only 2 patients showed a durable response in the absence of therapy. Disease recurrence in patients who continued therapy after HBeAg seroconversion was preceded by the development of resistance (80% of these patients), resistance only occurred in subjects given lamivudine monotherapy. In contrast, recurrence after treatment discontinuation or noncompliance was observed in all patients given nucleos(t)ide analogues. Conclusions: Induction of HBeAg seroconversion by nucleos(t)ide analogues is temporary in most patients with chronic HBV infection. Long-term continuation of nucleos(t)ide analogue treatment, irrespective of the occurrence of HBeAg seroconversion, appears to be necessary. © 2010 AGA Institute.