Antiviral therapy of influenza virus infections depends heavily on two viral neuraminidase (NA) inhibitors, oseltamivir (OSV) and zanamivir (ZNV). The efficacy of OSV is challenged by the development of viral resistance, while the clinical use of ZNV is limited by its poor pharmacokinetic profile and requirement for twice-daily intranasal administration. We have developed a novel NA inhibitor by conjugating ZNV to cholesterol. The ZNV-cholesterol conjugate showed markedly improved antiviral efficacy and plasma half-life compared with ZNV. Single-dose administration of the conjugate protected the mice from lethal challenges with wild-type or mutant H1N1 influenza viruses bearing an OSV-resistant H275Y-substitution. Mechanistic studies showed that the conjugate targeted the cell membrane and entered the host cells, thereby inhibiting the NA function and the assembly of progeny virions. The ZNV-cholesterol conjugate represents a potential new treatment for influenza infections with sustained effect. Cholesterol conjugation may be an effective strategy for improving the pharmacokinetics and efficacy of other small-molecule therapeutics.