The objective of the present study was to examine the association between cardiorespiratory fitness (CRF), insulin sensitivity and intrahepatic lipid content (IHL) measured by magnetic resonance spectroscopy. To perform this study a total of 138 overweight to obese asymptomatic subjects [age=43.6±8.9 years, body mass index (BMI)=33.8±4 kg/m2] were recruited. The data reported a positive association between CRF and insulin sensitivity. However, this relationship was lost after adjustment for intrahepatic fat content and maintained after controlling for total body fat, abdominal subcutaneous, abdominal intra-abdominal (visceral), and intramyocellular (IMCL) fat. A multivariate analysis revealed that IHL content was a stronger predictor of insulin sensitivity than intra-abdominal fat mass or IMCL content and independent of sex, age, or BMI. Moreover, in subjects with the highest CRF tertile, insulin sensitivity was unaffected by intra-abdominal adipose tissue mass or total body fat. In contrast, insulin sensitivity was consistently impaired in subjects with increased IHL regardless of physical fitness. These findings indicate that the relationship between CRF and insulin sensitivity appears to be mediated by hepatic lipid content.