There is no report on the gene expression profile of retinoblastoma (Rb). We analyzed the gene expression profile of Rb by the microarray technique. One thousand four genes were upregulated and 481 genes were downregulated. Microarray data were confirmed by semiquantitative RT-PCR for 5 genes in Rb samples: CDC25A, C17orf75, ERBB3, LATS2, and CHFR. Clusters of differentially expressed genes were identified on chromosomes 1, 16, and 17. Based on the expression profile, we hypothesized that the PI3K/AKT/mTOR (insulin signaling) pathway might be dysregulated in Rb. Our semiquantitative RT-PCR analysis of the PIK3CA, AKT1, FRAP1, and RPS6KB1 genes in Rb samples supported this hypothesis. We suggest that known inhibitors of this pathway could be evaluated for the treatment of Rb.