Early life stress is associated with long-term and pervasive adverse effects on neuroendocrine development, affecting normal cognitive and emotional development. Experimental manipulations like environmental enrichment (EE) may potentially reverse the effects of early life stress induced by maternal separation (MS) paradigm in rodents. However, the functional brain networks involved in the effects of EE after prolonged exposure to MS have not yet been investigated. In order to evaluate possible changes in brain functional connectivity induced by EE after MS, quantitative cytochrome c oxidase (CCO) histochemistry was applied to determine regional brain oxidative metabolism in adult male rats. Unexpectedly, results show that prolonged MS during the entire weaning period did not cause any detrimental effects on spatial learning and memory, including depressive-like behavior evaluated in the forced-swim test, and decreased anxiety-like behavior. However, EE seemed to alter anxiety- and depression-like behaviors in both control and MS groups, but improved spatial memory in the latter groups. Analysis of brain CCO activity showed significantly lower metabolic capacity in most brain regions selected in EE groups probably associated with chronic stress, but no effects of MS on brain metabolic capacity. In addition, principal component analysis of CCO activity revealed increased large-scale functional brain connectivity comprising at least three main networks affected by EE in both MS and control groups. Moreover, EE induced a pattern of functional brain connectivity associated with stress and anxiety-like behavior as compared with non-enriched groups. In conclusion, EE had differential effects on cognition and emotional behavior irrespective of exposure to MS. In view of the remarkable effects of EE on brain function and behavior, implementation of rodent housing conditions should be optimized by evaluating the balance between scientific validity and animal welfare.