The IL6 family of cytokines, including IL6 and leukemia-inhibitory factor (LIF), are induced during inflammation and are also expressed in many types of cancer where they play an important role in tumor development. IL6 family cytokines mainly activate the JAK-STAT3 pathway via the coreceptor, gp130, and IL6 is known to activate the Src family kinase (SFK)-Yes-associated protein (YAP) pathway. The current study investigated the role of autocrine LIF in human esophageal squamous cell carcinoma (ESCC) that highly expresses LIF. knockdown had various effects on cancer cells, including profound changes in gene expression, suppression of cell proliferation, migration/invasion and sphere formation, and induction of apoptosis. Similar to IL6, LIF activated the SFK-YAP pathway as well as the JAK-STAT3 pathway. LIF-induced YAP activation was more important for cancer cell proliferation than LIF-induced STAT3 activation, and concomitant YAP and STAT3 activation completely compensated for the role of LIF in human ESCC growth. We also confirmed that SFK activation and LIF expression were correlated with YAP activation in human ESCC clinical samples. Furthermore, simultaneous inhibition of the SFK-YAP and JAK-STAT3 pathways in human ESCC cells was more effective at suppressing cell proliferation than single inhibition, and autocrine LIF signaling promoted human ESCC growth . Therefore, the LIF-SFK-YAP axis may represent a new therapeutic target for human ESCC. IMPLICATIONS: Autocrine LIF signaling promotes human ESCC progression via SFK-dependent YAP activation and is a new potential target of treatment for human ESCC.