Two experiments were carried out during two successive years 2014 and 2015 at the period from July to October in the experimental apiary of Faculty of Agriculture, Benha University, Egypt to study the effect of honeybee (Apis mellifera L.) feeding with pollen substitute on bee venom production. As well as, the effect of venom gathering during various periods of the day on the number of dead bees and worker's brood rearing. After that, venom gland was microscopy examined and the collected venom was HPLC analyzed. Results indicated that the highest average of collected bee venom was 0.17 and 0.20 g/colony in July at 2014 and 2015, respectively. The highest venom amounts were collected at 7-9 pm. In addition, the higher mean numbers of dead bee’s (18.7 and 24.0 dead bee/colony) were observed in October and August of 2014 and 2015 year, respectively. Venom gathering effect on worker’s sealed brood area so it increased in control colonies by 4.0 and 13.0 % more than the colonies used for collection venom at different day times during 2014 and 2015, respectively. Results also showed that venom gland parameters were affected by feeding honeybee on pollen substitute. From HPLC analysis of the collected bee venom, results showed that the main components of venom were increased by feeding honey bee colonies on pollen substitute. The main components were melittin (48.7%), phospholipase A2 (16.4%) and apamin (2.0%).