Recent studies revealed a causal relationship between Toll-like receptors (TLRs) and lipid droplet biogenesis. Interestingly, it has been reported before that nanomaterials (NMs) were capable to modulate TLRs, but it remains unclear if NMs could affect lipid levels via TLR signaling pathways. In this study, we investigated the influences of airway exposure to graphene oxide (GO) on TLR3 signaling pathways and lipid levels in mouse livers. Intratracheal instillation of GO (0.1, 1, and 5 mg/kg, once a day, totally 5 days) induced inflammatory cell infiltrations as indicated by hematoxylin-eosin (H&E) staining and fibrosis as indicated by Masson staining in lungs, accompanying with decreased TLR3 proteins. Consistently, a TLR3-regulated anti-virus protein, namely interferon induced protein with tetratricopeptide repeats 1 (IFIT1), as well as two TLR3-regulated lipid proteins, namely radical S-adenosyl methionine domain containing 2 (RSAD2) and perilipin 2 (PLIN2), were decreased in lungs. The protein levels of interferon-β in serum were also decreased. In livers, GO exposure induced disorganization of liver cells but not fibrosis. In agreement with the trends observed in lungs, TLR3, IFIT1, RSAD2, and PLIN2 proteins were decreased in livers. As a possible consequence, GO exposure dose-dependently decreased lipid levels in livers as indicated by oil red O and BODIPY 493/503 staining. We concluded that airway exposure to GO decreased anti-virus responses and lipid levels in mouse livers via the suppression of TLR3.