(Abstract) Living organisms detect seasonal changes in day length (photoperiod) [1-3] and alter their physiological functions accordingly to fit seasonal environmental changes. TSHbeta, induced in the pars tuberalis (PT), plays a key role in the pathway that regulates vertebrate photoperiodism [4, 5]. However, the upstream inducers of TSHbeta expression remain unknown. Here we performed genome-wide expression analysis of the PT under chronic short-day and long-day conditions in melatonin-proficient CBA/N mice, in which the photoperiodic TSHbeta expression response is preserved . This analysis identified "short-day" and "long-day" genes, including TSHbeta, and further predicted the acute induction of long-day genes by late-night light stimulation. We verified this by advancing and extending the light period by 8 hr, which induced TSHbeta expression within one day. In the following genome-wide expression analysis under this acute long-day condition, we searched for candidate upstream genes by looking for expression that preceded TSHbeta's, and we identified the Eya3 gene. We demonstrated that Eya3 and its partner Six1 synergistically activate TSHbeta expression and that this activation is further enhanced by Tef and Hlf. These results elucidate the comprehensive transcriptional photoperiodic response in the PT, revealing the complex regulation of TSHbeta expression and unexpectedly rapid response to light changes in the mammalian photoperiodic system.