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Norovirus, the leading cause of gastroenteritis worldwide, is a non-enveloped virus whose tropism is determined in part by the expression patterns of entry receptors. However, the contribution of cellular lipids to viral entry is not well understood. Here, we determined that the asymmetrical distribution of lipids within membrane bilayers is required for murine norovirus (MNV) replication. Specifically, TMEM30a, an essential subunit of lipid flippases, is required for MNV replication in vitro. Disruption of TMEM30a in mouse intestinal epithelial cells prevents persistent, enteric infection by MNV in vivo. Mechanistically, TMEM30a facilitates MNV binding and entry. Surprisingly, exoplasmic phosphatidylserine (PS), a typical marker of dying cells, does not inhibit MNV infection. Rather, TMEM30a maintains a lipid-ordered state that impacts membrane fluidity that is necessary for the low affinity, high avidity binding of MNV to cells. Our data provides a new role for lipid asymmetry in promoting non-enveloped virus infection in vitro and norovirus persistence in vivo.