Acute lung injury (ALI) is featured by intensive inflammatory responses causing significant morbidity and mortality. Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), induced by interferon (IFN), has been discovered to modulate viral infection and cell apoptosis and inhibit the production of pro-inflammatory cytokines. However, it’s role and mechanism in ALI remain unclear and need to be explored furtherly. Here, we discovered that IFIT1 decreased the expression of TNF-α, IL-1β and IL-6 in mouse-derived macrophage cells (MH-S) and alleviated apoptosis of murine lung epithelial cells (MLE-12) induced by MH-S cell supernatant, contributing to anti-inflammatory and antiapoptotic effects in vitro and in vivo. Moreover, RNA sequencing analysis (RNA-seq) showed that inflammatory chemokine CC motif chemokine ligand 5 (CCL5) partially eliminated the protective effects of IFIT1 and promoted the expression of inflammatory cytokines TNF-α, IL-1β and IL-6 by CCL5-p65NF-κB signaling pathway. This study demonstrated that IFIT1 attenuated ALI-associated inflammation and cell apoptosis by regulating the CCL5-p65NF-κB signaling pathway. These findings are of great significance for the treatment of lung injury.