Breaking up prolonged sitting has been beneficially associated with cardiometabolic risk markers in both observational and intervention studies. We aimed to define the acute transcriptional events induced in skeletal muscle by breaks in sedentary time. Overweight/obese adults participated in a randomized three-period, three-treatment crossover trial in an acute setting. The three 5-h interventions were performed in the postprandial state after a standardized test drink and included seated position with no activity and seated with 2-min bouts of light- or moderate-intensity treadmill walking every 20 min. Vastus lateralis biopsies were obtained in eight participants after each treatment, and gene expression was examined using microarrays validated with real-time quantitative PCR. There were 75 differentially expressed genes between the three conditions. Pathway analysis indicated the main biological functions affected were related to small-molecule biochemistry, cellular development, growth and proliferation, and carbohydrate metabolism. Interestingly, differentially expressed genes were also linked to cardiovascular disease. For example, relative to prolonged sitting, activity bouts increased expression of nicotamide N-methyltransferase, which modulates anti-inflammatory and antioxidative pathways and triglyceride metabolism. Activity bouts also altered expression of 10 genes involved in carbohydrate metabolism, including increased expression of dynein light chain, which may regulate translocation of the GLUT-4 glucose transporter. In addition, breaking up sedentary time reversed the effects of chronic inactivity on expression of some specific genes. This study provides insight into the muscle regulatory systems and molecular processes underlying the physiological benefits induced by interrupting prolonged sitting.