Avian beta-defensins (avBDs) play significant roles in the innate immune system. The aim of this study was to identify immunoreactive (ir) avBDs proteins in the hen ovarian follicles and the changes in their localization with follicular growth. The ovarian follicles at different growth stages, namely the largest (171), second and third largest (F2 and F3), prehierarchal small yellow and cortical follicles, were collected. The presence of ir-avBD-8, -10, and -12 were examined by immunohistochemistry and Western blot. The three ir-avBDs showed a similar pattern of immunostainings in the follicular tissues at different growth stages. In the granulosa cells, the ir-avBDs were identified in the cortical follicles, whereas their density was reduced in small yellow follicles. The granulosa cells of yellow follicles (F3-F1) showed dense immunolabelings. The interstitial cells showed a faint immunolabeling for avBD-12 but not for avBD-8 and -10 in the cortical follicles, whereas they were weakly stained in the small yellow follicles. Dense immunoreaction products were noticed in the theca interna cells of F3-F1 follicles. Western blot analysis showed a single band for each defensin. These results suggest that avBD-8, -10 and -12 proteins are expressed in the specific cells in the follicles, namely interstitial or theca interna cells and granulosa cells, where their amounts are likely increased with follicular growth. These avBDs may play significant roles in the host innate immune system in the follicles.