Objective To establish an immortalized human feeder cell line that efficiently supports the growth of undifferentiated human embryonic stem ( hES) cells. Methods The immortalized TWE3R cell line was established by stably expressing the human E-cadherin gene in the TW2R cell line. The numbers of hESC colonies on TWE3R feeder cells were compared with those on its parental cell lines. After H9 hES cells were cultured on TWE3R cells for 10 consecutive passages, pluripotency marker analysis, embryoid body ( EB) formation assays and teratoma formation assays were conducted. Results We succeeded in establishing the TWE3R human feeder cell line that showed higher efficiency to support the growth of hES cells than its parental cell line. TWE3R feeder cells supported the growth of H9 ESCs at low density ( 5 cells/ cm²). Additionally, after long-term culture on TWE3R feeders, H9 ESCs retained all undifferentiated characteristics, a normal karyotype and the potential to differentiate into derivatives of all three embryonic germ layers. Conclusions The immortalized TWE3R human feeder cell line is capable of supporting efficient growth of human ESCs.