In modern poultry production, hens are raised apart from roosters. This is a large change from their natural social structure. We compared the productivity, injuries, behavior, physiology, microbiome and transcriptome of hens housed with (R+) or without (R-) roosters to quantify the effects of this change in social structure. Hens were housed free-range from 70-280 days when 30 birds per treatment were assigned to battery cages until day 315 (R+C vs R-C) whilst 30 birds remained in the free-range pens (R+F vs R-F). Response to novel environment and novel object, behavioral time budgets, cecum microbiome, blood composition and transcriptomic sequencing of thigh muscle and spleen were performed. Hens housed without roosters had better survival, consumed less food, produced more eggs and had better feed conversion. When present, R+F hens clustered around the rooster and were less mobile in novel environment and object test. R+F hens possessed the richest microbiome and the presence of roosters resulted in differentially expressed genes related to muscle development, cellular processes, environmental information processing and immune function. Removing roosters from housing hens intensified the desirable characteristics favored by domestication probably operating by deprivation of mating behavior and reduced fear together with altered microbial and genetic functions.