Interleukin-6 (IL-6), through activation of the signal transducer and activator of transcription 3 (STAT3) and trefoil factor family 3 (TFF3), has been implicated in the promotion of mouse biliary epithelial cell (BEC) proliferation and migration. However, it is still unclear whether the IL-6/STAT3/TFF3 signaling had similar effects on human BECs. Here, we showed that exposure of human BECs to recombinant IL-6 resulted in STAT3 phosphorylation and increased the expression of TFF3 at both mRNA and protein levels. Moreover, inhibition of STAT3 using RNA interference significantly abrogated IL-6-induced TFF3 expression. In an in-vitro wound healing model, IL-6 facilitated human BEC migration. This promotion of cell migration by IL-6 was blocked when STAT3 was knocked down. Interestingly, the addition of exogenous TFF3 could rescue the cell migration defects caused by STAT3 silencing. In conclusion, our data indicate that STAT3 plays a critical role in IL-6-induced TFF3 expression in human BECs and the IL-6/STAT3/TFF3 signaling is involved in human BEC migration and wound healing.