The Toll-like receptor 9 (TLR9) plays a crucial role in both innate and adaptive immune responses against infection and danger signals. Stimulation of TLR9 has been linked to invasion in various cancer cells in vitro. The present study evaluated the expression of TLR9 in human esophageal cancer (EC) cells and normal and malignant esophageal squamous epithelium, and examined the association between TLR9 expression, clinicopathological variables, and EC patient outcome. We further characterized the direct effects of TLR9 agonist CpG oligonucleotides (CpG ODN) and inhibitor chloroquine (CQ), on the proliferation and invasion of EC cells in vitro. RT-PCR, western blot, flow cytometry and immunohistochemical analysis were used to determine the expression of TLR9 in EC cell line TE10, and 90 cases of esophageal squamous cell carcinoma, including 30 cases of adjacent esophageal epithelium. The TLR9 expression was compared with tumor size, location, grade, stage and proliferation. We found basal expression of TLR9 in TE10 cells. Esophageal carcinomas exhibited TLR9 expression that was positively associated with tumor size, location and TNM stage (P<0.05). CpG ODN significantly enhanced the invasion of TE10 cells, which could be abrogated by a TLR9 inhibitor CQ. CpG ODN led to activation of NF‑κB and enhanced expression of matrix metalloproteinase (MMP)-2, MMP-7 and cyclooxygenase-2 (COX-2) mRNA. Expression of TLR9 in EC suggests a role of TLR9 related to cell proliferation and differentiation. Our findings indicate that TLR9 may represent a novel therapeutic target in this disease.