Hypertonic saline (HTS) is useful in the management of intracranial hypertension and shock patients. The aim of this study was to investigate whether HTS enhances host defense in burn mice through the increase of Toll-like receptors (TLRs) and nuclear factor κB (NF-κB) activation. C57BL/6, TLR4, C3H/HeN, and C3H/HeJ (nonfunctional TLR4 mutant) mice underwent burn and were given 10 mL/kg HTS (7.5% NaCl, 1.28 mol/L), 10 mL/kg saline (154 mmol/L), or 80 mL/kg saline (154 mmol/L) at 8 h after burn. At 24 h after burn, mesenteric lymph nodes were harvested and assayed for bacterial translocation (BT). Next, animals received i.p. Escherichia coli challenge, and bacterial clearance was measured. Finally, peritoneal cells were isolated for assay of bacterial killing activity, phagocytic activity, apoptotic ratio, NF-κB DNA binding activity, and expression of TLR4, MyD88, p-Akt, pp38, macrophage inflammatory protein 2, and Bcl-xL. Hypertonic saline decreased BT in C57BL/6 and C3H/HeN mice but not in TLR4 mutant mice. Also, HTS increased bacterial clearance and bacterial killing activity and decreased apoptotic ratio of peritoneal cells from C57BL/6 and C3H/HeN mice but not TLR4 or C3H/HeJ mice. Finally, HTS increased NF-κB activity and expression of TLR4, MyD88, p-Akt, pp38, macrophage inflammatory protein 2, and Bcl-xL in C57BL/6 but not in TLR4 mice. Hypertonic saline increases bacterial clearance and bacterial killing activity and decreases thermal injury-induced BT in wild-type but not in TLR4 mutant mice. Given that HTS induces NF-κB activity and TLR4, MyD88, and pp38 expression but decreases the apoptosis of inflammatory cells, we conclude that HTS resuscitation enhances host defense against bacterial challenge and reduces apoptosis of inflammatory cells in burn mice by increasing TLR4 expression and NF-κB activation.