In the US more than 100 million people are living with diabetes or pre-diabetes. The economic burden caused by these conditions, including medical costs, is approximately $322 billion annually as of 2013. Conventionally, transgenic Escherichia coli has been the primary source of commercial insulin production, a process that requires extensive purification to ensure shelf stability and complete removal of contaminants. This study seeks to establish an alternative mode of insulin production using polyethelyne glycol (PEG) to transform the oyster mushroom, Pleurotus ostreatus, with the human insulin gene. P. ostreatus is a valuable target for genetic transformation due to its lack of endotoxins and fully sequenced genome. P. ostreatus was transformed using PEG with a plasmid containing the human insulin gene and a carboxin resistance gene. Transformed cells were selected using carboxin, extracted, and regenerated on plates composed of yeast extract, malt extract, and glucose (YMG). Integration of the human insulin gene in to the mushroom genome was confirmed through PCR analysis of the transformants. Successful PEG transformation of P.ostreatus offers a new avenue for insulin production, potentially diversifying the market and treatment options for diabetics.