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Adult males and females have markedly different body composition, energy expenditure, and have different degrees of risk for metabolic diseases. A major aspect of metabolic regulation involves the appropriate storage and disposal of glucose and fatty acids. The use of sophisticated calorimetry, tracer, and imaging techniques have provided insight into the complex metabolism of these substrates showing that the regulation of these processes varies tremendously throughout the day, from the overnight fasting condition to meal ingestion, to the effects of physical activity. The sexual dimorphism in substrate metabolism is most readily observed in how fatty acids are stored and mobilized. The objective of this review is to provide a comprehensive and critical summary of the reported sex-differences in the mobilization, oxidation and storage of fat and carbohydrate in adipose tissue and skeletal muscle. We will describe how adipose tissue lipolysis differs between sexes, and how this varies between fed, fasted and exercise conditions. We will also review what is known about endogenous and exogenous fatty acid storage in adipose tissue and muscle, as well as how oxidation compares between men and women in response to exercise. What has been learned about the cellular level regulation of these processes will be described. Although glucose metabolism exhibits fewer differences between men and women, we will also review the existing knowledge on this topic.