Objective: Transmembrane protein 166 (TMEM166) expression in esophageal squamous cell carcinoma (ESCC) and remote normal esophageal tissues was examined to assess any role in tumour biology. Methods: TMEM166 mRNA expression in 36 cases with ESCC (36 tumour samples, 36 remote normal esophageal tissue samples) was detected by RT-PCR. TMEM166 protein expression was analysed in paraffin-embedded tissue samples from the same cases by immunohistochemistry. Results: Semi-quantitative analysis showed TMEM166 mRNA expression in ESCCs to be significantly lower than in remote normal esophageal tissues ($0.759{pm}0.713$ vs. $2.622{pm}1.690$, P=0.014). TMEM166 protein expression was also significantly reduced (69.4% vs. 94.4%, P<0.01). Conclusion: TMEM166 mRNA and protein expression demonstrated significant reduction in ESCCs compared with remote esophageal tissues, albeit with no correlation with tumour size, differentiation, stage, and lymph node metastasis, suggesting a role in regulating autophagic and apoptotic processes in the ESCC.