BMI-1 (B lymphoma Mo-MLV insertion region 1 homolog) has been reported to be over-expressed in cell immortalisation and the epithelial-mesenchymal transition (EMT) of cancer cells. The aim of this study is to study the roles of BMI-1 in the human telomerase reverse transcriptase (hTERT)-induced immortalisation and EMT. In this study, hTERT+-OME cells and hTERT+-HaCaT cells were acquired by viral transduction of hTERT to primary cultured oral keratinocytes and HaCaT cells (skin epidermal cells). siRNA transduction was used for the inhibition of BMI-1 expression. RT-PCR and Western blots were performed to detect the expressions of twist, vimentin, BMI-1, hTERT and p16INK4a in these cell lines. EMT was assessed by immunohistochemistry (expressions of cytokertin & vimentin), Western blots (expressions of Twist, vimentin & E-cadherin) and RT-PCR (expression of Twist). The results indicated that hTERT+-OME cells and hTERT+-HaCaT cells underwent EMT spontaneously with high expression of Twist. p16INK4a was silenced in both hTERT-transduced cells but could be detected in HaCaT cells. Moreover, BMI-1 was highly expressed in hTERT+-OME and hTERT+-HaCaT cells but was negative in HaCaT cells. When the expression of BMI-1 was blocked by siRNA transduction, the proliferations of hTERT+-OME and hTERT+-HaCaT cells were inhibited and the mono-spheroid colony formation of these hTERT-transduced cells was decreased. In addition, the expression of p16INK4a was regained while the expressions of EMT markers (twist and vimentin) were down-regulated in these two BMI-1 blocking cell lines. To conclude, this study suggests BMI-1 expression plays a role in hTERT-induced immortalisation and EMT.