The transcription factor E2F1 may form a complex with RB and which may play a major role in promoting growth control. Small interfering RNAs are short, double-stranded RNA molecules that can target mRNAs with complementary sequence for degradation via a cellular process to induce gene silence through degradation of homologous transcripts. The experiments were designed to inhibit proliferation of human retinoblastoma cells by knockdown human transcription factor E2F1 expression level using RNA interference technology. Three different hairpin shRNA inserts were designed targeting human transcription factor E2F1. The three inserts were synthesized and subcloned into a shRNA espression vector pSilencer H1puro which contains the H1 RNA polymerase Ⅲ promoter. The three plasmids encoding E2F1 short hairpin were transfected into human retinoblastoma cells line HXO-Rb-44 and human liver cancer cell line SMMC-7721 in vitro. Real-time RT-PCR was used to measure E2F1 mRNA level. For function analysis, propidium iodide (PI)-stained DNA content method was used to identify cell cycle by flow cytometer. The selected shE2F1 were cloned into an adenovirus system and packaged to adenovirus. Retinoblastoma HXO-Rb-44-gfp cell line which has been marked with green fluorescent protein (GFP) were infected by Ad-shE2F1 for 48h, and Ad-shNegative was used as control. The infected cells were injected into anterior chamber of nude mice eyes. The eyes were investigated and the consecutive and dynamic photos of the condition of RB tumor′s growth were taken by the special fluorescent microscope. One in three RNAi expression plasmids delivery resulted in 83.5% suppression of E2F1 mRNA transcription level. The same shRNA showed 89% inhibition in entering S phase. In nude mice eye anterior chamber, Ad-shE2F1 infected tumor cells grew more slowly than the negative control. The results showed that efficacy of Ad-shE2F1 were helpful in the process of limiting the growth of retinoblastoma. RNAi gene therapy directed against the human transcription factor E2F1 is a new therapeutic approach to suppress retinoblastoma cells proliferation.