Humans and animals host diverse communities of microorganisms important to their physiology and health. Despite extensive sequencing-based characterization of host-associated microbiomes, there remains a dramatic lack of understanding of microbial functions. Stable-isotope probing (SIP) is a powerful strategy to elucidate the ecophysiology of microorganisms in complex host-associated microbiotas. Here, we suggest that SIP methodologies should be more frequently exploited as part of a holistic functional microbiomics approach. We provide examples of how SIP has been used to study host-associated microbes in vivo and ex vivo. We highlight recent developments in SIP technologies and discuss future directions that will facilitate deeper insights into the function of human and animal microbiomes.