Cold-inducible RNA-binding protein (CIRP), released into the circulation during sepsis, causes lung injury via an as yet unknown mechanism. Since endoplasmic reticulum (ER) stress is associated with acute lung injury (ALI), we hypothesized that CIRP causes ALI via induction of ER stress. To test this hypothesis, we studied the lungs of wild-type (WT) and CIRP knockout (KO) mice at 20 h after induction of sepsis by cecal ligation and puncture (CLP). WT mice had significantly more severe ALI than CIRP KO mice. Lung ER stress markers (BiP, pIRE1α, sXBP1, CHOP, cleaved caspase-12) were increased in septic WT mice, but not in septic CIRP KO mice. Effector pathways downstream from ER stress – apoptosis, NF-κB (p65), proinflammatory cytokines (IL-6, IL-1β), neutrophil chemoattractants (MIP-2, KC), neutrophil infiltration (MPO activity), lipid peroxidation (4-HNE), and nitric oxide (iNOS) – were significantly increased in WT mice, but only mildly elevated in CIRP KO mice. ER stress markers were increased in the lungs of healthy WT mice treated with recombinant murine CIRP, but not in the lungs of TLR4 KO mice. This suggests CIRP directly induces ER stress via TLR4 activation. In summary, CIRP induces lung ER stress and downstream responses to cause sepsis-associated ALI.