Prolactin, glucocorticoid and insulin signalling regulates platypus MLP and BLG milk protein gene expression in BME-UV1 cells. Transcription factors essential for the hormonal responsiveness are conserved in MLP and BLG promoters. Monotreme milk protein genes are hormonally regulated in the same way as distantly related marsupial and eutherian mammals. Lactogenic hormonal regulation of milk protein gene expression evolved at least 166 million years ago.