Transferring foreign genes into mushroom mediated by Agrobacterium tumefaciens is a standard technique in genetic engineering. Recombinant human insulin has been greatly used in the treatment of type I diabetes. The production of edible mushroom derived insulin should facilitate oral delivery. In this study we used the Agrobacterium tumefaciens mediated transformation method for the transfer and expression of the cholera toxin-B subunit (CTB) fused with human pro-insulin (Pins) in the edible oyster mushroom, Pleurotus ostreatus. The binary vector pCAMBIA1304 harboring CTB-Pins gene driven by the P. ostreatus gpd promoter was constructed and introduced into P. ostreatus via Agrobacterium tumefaciens mediated transformation. Optimization of gene transformation and tissue culture conditions is one of the most important issues in transgenic mushroom production. For attaining optimized conditions, the effects of co-cultivation conditions, Agrobacterium strains and the concentration of Agrobacterium cell suspensions were studied. Integration of CTB-Pins into mushroom genome was confirmed by PCR. The successful transformation to oyster mushroom suggests that the proposed modified transformation system is apparently useful for the production of human pro-insulin in these edible mushrooms.