MicroRNAs (miR) are small non-coding single-stranded RNA molecules, normally 22 nucleotides long. There is a large family of miR that control protein expression by binding to mRNAs, leading to their degradation. One important role of miR is in tumours where they may promote, via down-regulation of suppressor proteins, or prevention by down regulation of oncogenic proteins. In this study miR21, which has been shown to be involved in proliferation, metastasis and anti-apoptosis of tumour cells, was analysed in retinoblastoma. miR21 expression was increased in a human Rb cell line, HXO-RB44, and Rb tissue, compared to normal retinal tissue. Antisense-miR (22 nucleotides), or seed-targeting 8mer oligonucleotides, transfected into cells led to a significant decrease in proliferation, migration and colony-forming in vitro. Antisense miR downregulated miR21 levels while the 8mer oligonucleotide did not. To determine the pathways involved in the inhibition phosphorylation status of HXO-RB44 cells treated with anti-MiR21 was assessed. Levels of phosphorlyated signal proteins such as BAD and Akt were downregulated. The data supports a role for miR21 in retinoblastoma and that inhibition of this miR could have therapeutic value.