Background: Early diagnosis of esophageal squamous cell carcinoma (ESCC) is an important issue to improve the prognosis. HOX transcript antisense RNA (HOTAIR), a long noncoding RNA (lncRNA) expressed from the HOXC locus, has been recently revealed as an oncogenic regulator in ESCC. This study aimed to investigate whether serum HOTAIR is involved in the diagnosis of ESCC. Methods: In this study, we detected serum HOTAIR expression in 50 patients with ESCC (including 42 tumor resection and 8 without surgery) and 20 healthy volunteers to investigate the role of serum HOTAIR in ESCC using the quantitative real-time polymerase chain reaction (qRT-PCR) method. Results: Clinical data indicated that serum HOTAIR were correlated with TNM stage. The expression level of serum HOTAIR (0.189 +/- 0.010) was significantly higher in ESCC patients compared with that of healthy controls (0.055 +/- 0.008, P < 0.01). The ROC curve analysis yielded an area under the ROC curve (AUC) value of 0.793 (95% CI: 0. 692 to 0.895, P < 0.01). Also, the serum HOTAIR expression level decreased obviously in postoperative samples (one month after the surgery) compared to preoperative specimens. Moreover, there was a significant correlation between serum HOTAIR expression and the expression of HOTAIR in ESCC tissue according to Pearson correlation analysis. Conclusions: Our study, for the first time, demonstrated that serum HOTAIR might serve as a potential biomarker for the diagnosis of ESCC.