Gap junction intracellular communication (GJIC) allows the direct transport of small molecules between adjacent cells. We hypothesized that siRNAs in one hESC could inhibit target RNA expression in another hESC via GJIC. We co-cultured green fluorescent protein (GFP)-expressing ENVY hESC with non-GFP-expressing hESC, which had been transduced to stably express shRNA directed against GFP. We discovered that the GFP shRNA expressing hESC inhibited GFP expression in the adjacent GFP-expressing hESC in a dose-dependent manner. This downregulation of GFP expression in ENVY cells was not observed when the co-cultured cells had been transduced with a non-functional GFP shRNA that was mutated at two nucleotides or when the cells were incubated with the GJIC inhibitor, alpha-glycyrrhetinic acid (alpha-GA). We conclude that 21-23 bp double-stranded shRNA/siRNA oligonucleotides are able to move through gap junctions between hESCs and thus can affect gene expression in neighbouring hESC. This novel intercellular gene expression regulatory mechanism may offer new approaches to manipulation of hESC.