Skeletal muscles are highly plastic tissues capable dramatic remodeling in response to use, disuse, disease, and other factors. Growing evidence suggests that adipose tissues exert significant effects on the basic fiber-type composition of skeletal muscles. In the current study, we investigated the long-term effects of a high-fat diet and subsequent obesity on the muscle fiber types in C57 BLK/6J mice. Litters of mice were randomly assigned to either a high-fat diet or a control group at the time of weaning, and were maintained on this diet for approximately 1 year. Single fibers were harvested from the soleus and plantaris muscles, and fiber types were determined using SDS-PAGE. The high-fat diet mice were significantly heavier than the control mice (39.17 ± 2.7 g vs. 56.87 ± 3.4 g; P < 0.0003), but muscle masses were not different. In male mice, the high-fat diet was associated with a significantly lower proportion of slow, type I fibers in the soleus muscle (40.4 ± 3.5% vs. 29.33 ± 2.6%; P < 0.0165). Moreover, the proportion of type I fibers in the soleus of male mice was inversely proportional to the relative fatness of the male mice (P < 0.003; r (2) = 0.65), but no association was observed in female mice. In male mice, the decline in type I fibers was correlated with an increase in type I/IIA hybrid fibers, suggesting that the type I fibers were transformed primarily into these hybrids. The reported trends indicate that type I fibers are most susceptible to the effects of obesity, and that these fiber-type changes can be sex specific.