Microarray data suggested that the expression of aldehyde dehydrogenase 1 family member L1 (ALDH1L1) is reduced in hepatocellular carcinoma (HCC). However, the role of ALDH1L1 in HCC carcinogenesis has not been elucidated. In the present study, we investigated the expression of ALDH1L1 in HCC and evaluated its relationship with clinical features and prognosis of HCC patients. Total 112 tumor samples were collected from patients with HCC, who underwent radical hepatectomy. Out of the 112 samples, 16 paired HCC tumorous and corresponding adjacent nontumor tissues were analyzed by real-time quantitative RT-PCR (qRT-PCR) and Western blotting, and the other 96 HCC samples were detected by immunohistochemical method. The qRT-PCR assay showed that the mRNA level of ALDH1L1 was significantly reduced in tumorous tissues compared with the adjacent nontumorous tissues (P = 0.0007), and Western blotting indicated that protein level of ALDH1L1 also notably down-regulated in tumor tissues. Immunohistochemistry detection revealed that decreased ALDH1L1 expression was present in 56.3% (54/96) of HCC patients. Correlation analysis showed that ALDH1L1 expression was significantly correlated with histological differentiation (P = 0.001), HBsAg status (P = 0.024) and serum AFP (P = 0.001). Patients with low expression of ALDH1L1 had poorer prognosis than those with high expression (P = 0.008). Multivariate analysis showed that ALDH1L1 expression was an independent predictor of overall survival (HR, 0.349; 95% CI, 0.157-0.774; P = 0.01). The results in this study, for the first time, reveal that the mRNA and protein expressions of ALDH1L1 are significantly reduced in HCC tissues and its low protein expression is a new and potential prognostic marker for the survival of HCC patients.