5-Fluorouracil (5-FU) is widely used as an anti-cancer drug, but causes severe side effects. Controlled release systems could be useful to keep the concentration of 5-FU at a low level, so that the side effects can be reduced. The purpose of this research study was to formulate transdermal film loaded with 5-Fluorouracil by solution casting method using ethyl cellulose, HPMC E15 and HPMC K4M in various combinations and to investigate the effect of different polymer on drug permeation and other physicochemical characteristics of the film. The ex-vivo permeation study was carried out with rat skin along with in-vitro study using dialysis membrane and both revealed that HPMC E15can exhibit more sustained formulation than HPMC K4M.The hydrophilic polymer alone was incapable to form the film. Further, it was observed that the cumulative % release of the drug from the formulation was retarded with the addition of the hydrophobic polymer EC and increased on addition of the hydrophilic polymer. Again, between two grades of HPMC i.e., HPMC E15 and HPMC K4M, the polymer E15 provided slower rate of release than HPMC K4M. The formulation FF1 with EC showed lowest moisture up take (1.9%) and also lowest % of release (56.76%) in 7 hr than the other formulations. Further, formulation FF2 with combination of HPMC K4M and EC showed highest moisture up take (18.78%) and also highest % of release (96.53%) of drug in7 hr and reduced in respect of moisture uptake and rate of release with the admixture of HPMC E15 and we also observed that there exist a decrease in order of the above with the increase of the concentration of the same polymer HPMC E15. Stability study at intermediate and accelerated conditions according to the ICH (Q1A) specifications showed that all the formulations were found to be very stable. Hence, transdermal film of 5-FU thus formulated could be a promising alternative dosage form in cancer chemotherapy.