IL-27 is involved in inflammatory reactions. CXCL10 is an important chemokine contributing to airway inflammatory disease. In this study, we investigated whether IL-27 modulated the synthesis of CXCL10 in primary human lung fibroblasts (HLFs). HLFs were activated by IL-27 alone, or in combination with other cytokines. CXCL10 synthesis was measured by real-time PCR and ELISA. An examination of transcriptional regulation was performed via the transient transfection of promoter constructs, whereas mRNA stability was assessed by actinomycin D chase and real-time PCR. The underlying signaling pathways were studied by Western blotting and intracellular staining, using flow cytometry. Our results demonstrated that IL-27 induced and synergized with TNF-α to up-regulate CXCL10 mRNA and protein concentrations in a steroid-insensitive manner. This synergistic CXCL10 production was dependent on the transcriptional regulation of CXCL10 gene promoter activity and the enhanced stability of CXCL10 mRNA because of IL-27 and TNF-α, and this synergism was regulated by the activation of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-OH kinase (PI3K)-Akt dominantly, and in small part via NF-κB. Interestingly, IL-27 promoted the basal and enhanced TNF-α-induced phosphorylation of p38 MAPK and Akt, but not IκBα. Moreover, enhanced CXCL10 mRNA stability occurred via a p38 MAPK-dependent pathway. Finally, clinical analysis showed that IL-27 was detected in the bronchoalveolar lavage of patients with asthma, chronic obstructive pulmonary disease (COPD), and pulmonary tuberculosis (PTB), and increased IL-27 concentrations were correlated with increased CXCL10 concentrations in patients with COPD and PTB. Our findings suggest that IL-27 has the potential to amplify airway inflammation via the induction of CXCL10 from HLFs, in combination with TNF-α.