Microbial rewilding, whereby exposure to naturalistic environments can modulate or augment gut microbiomes and improve host-microbe symbiosis, is being harnessed as an innovative approach to human health, one that may also have significant value to animal care and conservation. To test for microbial rewilding in animal microbiomes, we used a unique population of wild-born ring-tailed lemurs (Lemur catta) that were initially held as illegal pets in unnatural settings and, subsequently, relocated to a rescue center in Madagascar where they live in naturalistic environments. Using amplicon and shotgun metagenomic sequencing of lemur and environmental microbiomes, we found multiple lines of evidence for microbial rewilding in lemurs that were transitioned from unnatural to naturalistic environments: A lemur’s duration of exposure to naturalistic settings significantly correlated with (a) increased compositional similarly to the gut communities of wild lemurs, (b) decreased proportions of antibiotic resistance genes that were likely acquired via human contact during pethood, and (c) greater covariation with soil microbiomes from natural habitats. Beyond the inherent psychosocial value of naturalistic environments, we find that actions, such as providing appropriate diets, minimizing contact with humans, and increasing exposure to natural environmental consortia, may assist in maximizing host-microbe symbiosis in animals under human care.