Exercise enhances mitochondrial biogenesis in skeletal muscle. Increased mitochondrial function and content can contribute to the improvement in skeletal muscle function and the benefits of exercise by increasing the response to energy demands. The effect of standardized Kaempferia parviflora extract (KPE) on exercise performance was accessed in L6 myotubes and C57BL/6J mice. KPE significantly activated peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and increased mitochondrial density in L6 myotubes. KPE also upregulated the expression of transcription factors for mitochondrial biogenesis (estrogen-related receptor-α [ERRα], nuclear respiratory factor-1 [NRF-1], and mitochondrial transcription factor A [Tfam]) through activation of PGC-1α in L6 myotubes. In vivo models including normal diet mice and high-fat diet obese mice showed that KPE effectively enhanced running endurance and increased the skeletal muscle weight/body weight ratio. Furthermore, these observations were associated with a significant upregulation of mitochondrial biogenesis regulatory genes in skeletal muscle tissue. KPE enhanced the protein expression of the sirtuin 1 (SIRT1)/adenosine monophosphate (AMP)-activated protein kinase (AMPK)/PGC-1α/peroxisome proliferator-activated receptor-δ (PPARδ) signaling pathway components in vitro and in vivo, acting as an exercise metabolism regulator. These results suggest that KPE has the potential to enhance exercise performance through mitochondrial biogenesis and the SIRT1/AMPK/PGC-1α/PPARδ signaling pathways.