Insulin receptor isoform A (IR-A) is a fetal insulin receptor isoform that is overexpressed in cancer. We investigated whether insulin-like growth factor (IGF)-II may elicit a different gene expression response from insulin in cells expressing only IR-A and lacking IGF-I receptor (R-/IR-A cells). Cells were stimulated with either insulin or IGF-II (at 0.5, 3, and 8 h), and global gene expression was studied by microarray technology. Results were validated by quantitative real-time PCR. We found that 214 transcripts were similarly regulated by insulin and IGF-II, whereas 45 transcripts were differentially regulated. Of these 45 genes, 18 were responsive to only one of the two ligands (12 to insulin and 6 to IGF-II). Twenty-seven transcripts were regulated by both ligands but with a significant difference at at least one time point. IGF-II was a more potent regulator than insulin for these genes. In conclusion, insulin and IGF-II, acting via the same receptor (IR-A), may differentially affect gene expression in cells. These findings provide a molecular basis in clarifying the biological role of IR-A in embryonic/fetal life and in cancer.