Zebrafish larvae spend more time in brightly illuminated area when placed in a light/dark testing environment. Here we report that the anxiolytic drugs lorazepam and diazepam increased the time larval fish spent in the dark compartment in the light/dark test. Lorazepam did not affect the visual induced optokinetic response of larval fish. Gene expression levels of c-fos and crh were significantly increased in the hypothalamus of fish larvae underwent light/dark choice behavior, whilst lorazepam treatment alleviated the increased c-fos and crh expressions. Furthermore, we found estrogen receptor β gene expression level was increased in fish larvae underwent light/dark choice. We next examined effects of estrogen receptor modulators (estradiol, BPA, PHTPP, and WAY-200070) in the light/dark test. We identified WAY-200070, a highly selective ERβ agonist significantly altered the light/dark choice behavior of zebrafish larvae. Further investigation showed WAY-200070 treatment caused a reduction of crh expression level in the hypothalamus, suggesting activation of ERβ signaling attenuate the stress response. Interestingly, WAY-200070 treatment caused marked increase of c-fos expression in the habenula of fish larvae underwent behavior test. These results suggest WAY-200070 activation of ERβ mediated signaling may regulate anxiety related behavior in zebrafish through modulation of neuronal activity in habenula.