Rapid and effective control of bacterial infection is critical for the treatment of bacterial sepsis. CXCL14 (CXC motif ligand 14) is an important chemokine involved in infection and immunity, which can bind to CXCR4. However, the contribution of the CXCL14/CXCR4 chemokine axis to bacterial clearance in sepsis remains unknown. Here, the impact of CXCL14/CXCR4 blockade or CXCL14 administration on sepsis was assessed using murine and cell models, as well as human samples. CXCL14 protein concentrations were elevated in mice after cecal ligation and puncture (CLP)-induced sepsis. In vivo, CXCL14 blockade using anti-CXCL14 antibody or CXCL14 knockdown by adeno-associated virus carrying–CXCL14 shRNA significantly increased mortality and bacterial burden, which was paralleled by significantly decreased macrophage influx and M2 macrophage polarization at the site of infection after CLP. Therapeutic administration of CXCL14 improved mortality and bacterial clearance after CLP in a CXCR4-dependent manner, and macrophages, but not neutrophils, were important for the protective effect of CXCL14 in sepsis. In vitro, CXCL14 directly enhanced bacterial phagocytosis and killing of macrophages, and it also increased phagosome formation and reactive oxygen species production in macrophages. Furthermore, inhibiting the activation of PI3K/Akt and NF-κB signaling pathways, but not STAT1 (signal transducer and activator of transcription 1), abrogated the enhanced antibacterial effects of CXCL14 on macrophages. Finally, circulating CXCL14 concentrations were significantly upregulated in patients with sepsis. CXCL14 could enhance bacterial phagocytosis and killing in human monocyte-derived macrophages, which was dependent on CXCR4. Therefore, our results indicate a previously undescribed role of the CXCL14/CXCR4 axis and suggest CXCL14 as a potential adjunct therapy in bacterial sepsis.