Moderate-intensity exercise at the lactate threshold (LT) is considered to be a safe and effective training regimen for improving metabolic syndrome. The aim of the current study was to investigate the effects of moderate exercise performed at the LT on skeletal muscle gene expression. 6 healthy men participated in cycle ergometer training at LT, 60 min/d, 5 d/wk for 12 wks. Muscle samples were collected after 5 d of training, and then 2 d after training at wks 6 and 12. Quantitative real-time PCR analysis revealed that the expression of peroxisome proliferator activated receptor co-activated 1alpha was significantly increased at 1 h after the training session on day 5. Moreover, using serial analysis gene expression, we found that moderate training for 6 and 12 wks simultaneously induced the expression of a number of metabolic genes involved in the TCA cycle, beta-oxidation, and electron transport. Furthermore, several genes encoding antioxidant enzymes and contractile apparatus were induced. The expression levels of 233 novel transcripts were also altered in response to moderate exercise. Thus, moderate training at the LT is a sufficient stimulus to induce the expression of numerous genes implicated in the development of metabolic syndrome, transcripts involved in the contractile apparatus, and novel transcripts.