Altered gut microbial diversity has been associated with several chronic disease states, including heart failure. Stimulation of the vagus nerve, which innervates the heart and abdominal organs, is proving to be an effective therapeutic in heart failure. We hypothesized that cervical vagus nerve stimulation (VNS) could alter fecal flora and prevent aberrations observed in fecal samples from heart failure animals. To determine whether microbial abundances were altered by pressure overload (PO), leading to heart failure and VNS therapy, a VNS pulse generator was implanted with a stimulus lead on either the left or right vagus nerve before creation of PO by aortic constriction. Animals received intermittent, open-loop stimulation or sham treatment, and their heart function was monitored by echocardiography. Left ventricular end-systolic and diastolic volumes, as well as cardiac output, were impaired in PO animals compared with baseline. VNS mitigated these effects. Metagenetic analysis was then performed using 16S rRNA sequencing to identify bacterial genera present in fecal samples. The abundance of 10 genera was significantly altered by PO, 8 of which were mitigated in animals receiving either left- or right-sided VNS. Metatranscriptomics analyses indicate that the abundance of genera that express genes associated with ATP-binding cassette transport and amino sugar/nitrogen metabolism was significantly changed following PO. These gut flora changes were not observed in PO animals subjected to VNS. These data suggest that VNS prevents aberrant gut flora following PO, which could contribute to its beneficial effects in heart failure patients.