Embryonic stem (ES) cells can maintain self-renewal and differentiate into all three embryonic germ layer derivatives. The regulatory network of key transcription factors including Oct4, Nanog, and Sox2 plays a crucial role in maintaining the pluripotency of ES cells. Dissection of these transcriptional regulators has provided critical insights into mechanisms underlying the self-renewal and early differentiation of ES cells. Here, we identified a highly differentially expressed EST between human ES (hES) cells and 7-day embryoid bodies (EBs) by microarray analysis. By EST assembly, 5'-RACE and Northern blot, a novel hES cells related gene (named as HESRG) with two different transcripts in size of 1.2 and 3.1 kb was successfully cloned. Further RT-PCR and real-time RT-PCR (qPCR) results showed that HESRG was highly expressed in undifferentiated hES cells, but not expressed in all kinds of normal human abortive fetal tissues or human adult testis. The expression of HESRG was repressed upon differentiation of hES cells, in vitro and in vivo. Moreover, the transcription factor Oct4 binding site was found in the putative promoter region of HESRG. The above suggested an important role for the novel HESRG gene in supporting the undifferentiated state or self-renewal of hES cells.