Cellular tropism during persistent viral infection is commonly conferred by the interaction of a viral surface protein with a host receptor complex. Norovirus, the leading global cause of gastroenteritis, can be persistently shed during infection, but its in vivo cellular tropism and tropism determinants remain unidentified. Using murine norovirus (MNoV), we determine that a small number of intestinal epithelial cells (IECs) serve as the reservoir for fecal shedding and persistence. The viral non-structural protein NS1, rather than a viral surface protein, determines IEC tropism. Expression of NS1 from a persistent MNoV strain is sufficient for an acute MNoV strain to target IECs and persist. In addition, interferon-lambda (IFN-λ) is a key host determinant blocking MNoV infection in IECs. The inability of acute MNoV to shed and persist is rescued in Ifnlr1−/− mice, suggesting that NS1 evades IFN-λ-mediated antiviral immunity. Thus, NS1 and IFN-λ interactions govern IEC tropism and persistence of MNoV. The in vivo tropism of norovirus, a gastrointestinal pathogen, is unknown. Lee et al. discover that a small number of intestinal epithelial cells are a reservoir for persistent murine norovirus. This tropism is regulated by viral protein NS1 and host IFN-lambda, findings that provide critical insight into norovirus pathogenesis.