This study was carried out to determine the effects of in ovo pollen extract injection on growth parameters, ileal histomorphology, and caecal microflora of fasted broiler chicks. In this experiment, 2×2 factorial experimental design was used. One d old, 120 healthy broiler chicks were allocated to 4 treatment groups and 6 replicates (5 mixed sex chicks allocated each replicates). Treatment groups were: A) Pollen extract injection and 24 h fasting (P24); B) Pollen extract injection and 48 h fasting (P48), C) Control, no injection 24 h fasting (C24) and D) Control, no injection 48 h fasting (C48). The experiment lasted 21 days. Live weight, feed consumption, feed conversion ratio were recorded weekly. Ileal histomorphology, caecal microbiota, organ weight were recorded at 21th days of experiment. In ovo pollen extract injection did not affect hatchability rate. At the end of 21 days, in ovo pollen extract injection did not affect feed intake, live weight gain, feed conversion ratio, inner organ development and ileal villi width irrespective to fasting 24 h and 48 h. In ovo pollen extract injection increased ileal villi length, caecal lactic acid bacteria and Saccharomyces Cerevisiae count, decreased caecal Enterobactericaea count. In conclusion, in ovo pollen extract injection can be applied for broiler eggs to improve weight gain, better digestion and gut health.