Abstract： Background::Epithelial to mesenchymal transition (EMT) is a key process in determining distant metastasis and intra-hepatic dissemination of hepatocellular carcinoma (HCC). Follistatin ( FST) family members are considered to be an attractive therapeutic targets and prognostic indicators in cancers. As a derivative of FST, Follistatin Like 5 ( FSTL5) may play a similar role in HCC cells. This study aimed to investigate the expression and function of FSTL5 in HCC and its role in EMT. Methods::FSTL5, E-cadherin and vimentin in HCC, and paracancerous tissues were detected by immunohistochemistry. Correlation of FSTL5 expression with overall survival was assessed. The proliferation and invasion of HCC cell lines SK-Hep1 and MHCC-LM3 were analyzed by cell counting kit-8 and Transwell assays. The expression of FSTL5, E-cadherin, and vimentin in HCC cells was examined by polymerase chain reaction and Western blot analysis. T-test was used to analyze the difference in proliferation and invasion ability between groups. The Spearman rank correlation test was used to detect the correlation between the expression of FSTL5 and E-cadherin or vimentin. Results::The expression of FSTL5 in HCC was lower than that in paracancerous tissues (9.97% vs. 82.55%, χ2 = 340.15, P < 0.001). Patients with high FSTL5 expression had a better prognosis ( χ2= 8.22, P= 0.004) and smaller tumor diameter ( χ2 = 45.52, P < 0.001), less lymph node metastasis ( χ2 = 5.58, P= 0.02), earlier tumor node metastasis stage ( χ2 = 11.29, P= 0.001), a reduced number of tumors ( χ2 = 5.05, P= 0.02), lower alpha-fetoprotein value ( χ2 = 24.36, P < 0.001), more probability of hepatitis carrying ( χ2 = 40.9, P < 0.001), and better liver function grade ( χ2 = 5.21, P = 0.02). Immunohistochemistry showed that FSTL5 expression in HCC tissues was positively correlated with E-cadherin expression ( r = 0.38, P < 0.001) and negatively correlated with vimentin expression ( r = -0.385, P < 0.001). Furthermore, over-expression of FSTL5 up-regulated the expression of E-cadherin and down-regulated the expression of vimentin in SK-Hep1 (negative control [NC] vs. FSTL5-interfering group [Lv- FSTL5]: E-cadherin [ t= 45.03, P < 0.001], vimentin [ t= 67, P < 0.001]) and MHCC-LM3 (NC vs. Lv- FSTL5: E-cadherin [ t = 50, P < 0.001], vimentin [ t = 72.75, P < 0.001]) cells at mRNA level. The same as protein level. In addition, the over-expression of FSTL5 inhibited the proliferation (NC vs. Lv- FSTL5: SK-Hep1, 3 d [ t = 7.324, P = 0.018], 4 d [ t = 6.23, P = 0.021], 5 d [ t= 10.21, P= 0.003]; MHCC-LM3, 3 d [ t= 4.32, P= 0.037], 4 d [ t= 7.49, P= 0.012], 5 d [ t= 9.3661, P = 0.009]) and invasion (NC vs. Lv- FSTL5: SK-Hep1, t= 21.57, P < 0.001; MHCC-LM3, t= 18.04, P < 0.001) of HCC cells. Conclusions::Down-regulation of FSTL5 may contribute to EMT of HCC, and FSTL5 is a potential target in the treatment of HCC.