To examine the long-term infectivity of human norovirus in water, 13 study subjects were challenged at different time points with groundwater spiked with the prototype human norovirus, Norwalk virus. Norwalk virus spiked in groundwater remained infectious after storage at room temperature in the dark for 61 days (the last time point tested). The Norwalk virus-seeded groundwater was stored for 1,266 days and analyzed, after RNase treatment, by reverse transcription-quantitative PCR (RT-qPCR) to detect Norwalk virus RNA contained within intact capsids. Norwalk virus RNA within intact capsids was detected in groundwater for 1,266 days, with no significant log10 reduction throughout 427 days and a significant 1.10-log10 reduction by day 1266. Purified Norwalk virus RNA (extracted from Norwalk virus virions) persisted for 14 days in groundwater, tap water, and reagent-grade water. This study demonstrates that Norwalk virus in groundwater can remain detectable for over 3 years and can remain infectious for at least 61 days. (ClinicalTrials.gov identifier NCT00313404.