Esophageal cancer is the eighth most common cancer in the world and has an extremely dismal prognosis, with a 5-year survival of less than 20%. Current treatment options are limited, and thus identifying new molecular targets and pathways is critical to derive novel therapies. Worldwide, more than 90% of esophageal cancers are esophageal squamous cell cancer (ESCC). Previously, we identified that Krüppel-like factor 5 (KLF5), a key transcriptional regulator normally expressed in esophageal squamous epithelial cells, is lost in human ESCC. To examine the effects of restoring KLF5 in ESCC, we transduced the human ESCC cell lines TE7 and TE15, both of which lack KLF5 expression, with retrovirus to express KLF5 upon doxycycline induction. When KLF5 was induced, ESCC cells demonstrated increased apoptosis and decreased viability, with up-regulation of the proapoptotic factor BAX. Interestingly, c-Jun N-terminal kinase (JNK) signaling, an important upstream mediator of proapoptotic pathways including BAX, was also activated following KLF5 induction. KLF5 activation of JNK signaling was mediated by KLF5 transactivation of two key upstream regulators of the JNK pathway, ASK1 and MKK4, and inhibition of JNK blocked apoptosis and normalized cell survival following KLF5 induction. Thus, restoring KLF5 in ESCC cells promotes apoptosis and decreases cell survival in a JNK-dependent manner, providing a potential therapeutic target for human ESCC.