Poor aerobic fitness is linked to nonalcoholic fatty liver disease and increased all-cause mortality. We previously found that low capacity running (LCR) rats fed acute high fat diet (HFD; 45% kcal from fat) for 3 days resulted in positive energy balance and increased hepatic steatosis compared with high aerobically fit, high capacity running (HCR) rats. Here, we tested the hypothesis that poor physiological outcomes in LCR rats following acute HFD feeding are associated with alterations in cecal microbiota. LCR rats exhibited greater body weight, feeding efficiency, 3 d body weight change, and liver triglycerides after acute HFD feeding compared with HCR rats. Furthermore, LCR rats exhibited reduced expression of intestinal tight junction proteins compared with HCRs. Cecal bacterial 16S rDNA revealed that LCR rats had reduced cecal Proteobacteria compared with HCR rats. Microbiota of HCR rats had greater, Desulfovibrionaceae, unassigned genera within Desulfovibrionaceae, suggesting increased reduction of endogenous mucins and proteins. While acute HFD feeding reduced Firmicutes in both strains, short-chain fatty acid-producing Phascolarctobacterium was reduced in LCR rats. In addition, Ruminococcae and Ruminococcus were negatively correlated with energy intake in the LCR/HFD rats. Predicted metagenomic function suggests that LCR rats have a greater metabolic capacity to metabolize carbohydrate and energy compared with HCR rats. Overall, these data suggest that the populations and metabolic capacity of the microbiota in low aerobically fit LCR rats may contribute to their susceptibility to acute HFD-induced hepatic steatosis and poor physiologic outcomes.