Background and Objectives: Hyaluronan preserves the proliferation and differentiation potential of mesenchymal stem cells. Supplementation of low-concentration hyaluronan (SHA) in stem cells culture medium increases its proliferative rate, whereas coated-surface hyaluronan (CHA) maintains cells in a slow-proliferating mode. We have previously demonstrated that in CHA, the metabolic proliferative state of stem cells was influenced by upregulating mitochondrial biogenesis and function. However, the effect of SHA on stem cells’ energetic status remains unknown. In this study, we demonstrate the effect that low-concentration SHA at 0.001 mg/ml (SHA0.001) and high-concentration SHA at 5 mg/ml (SHA5) exert on stem cells’ mitochondrial function compared with CHA and noncoated tissue culture surface (control). Methods: and Results: Fast-proliferating human placenta-derived mesenchymal stem cells (PDMSCs) cultured on SHA0.001 exhibited reduced mitochondrial mass, lower mitochondrial DNA copy number, and lower oxygen consumption rate compared with slow-proliferating PDMSCs cultured on CHA at 5.0 (CHA5) or 30 μg/cm2 (CHA30). The reduced mitochondrial biogenesis observed in SHA0.001 was accompanied by a 2-fold increased ATP content and lactate production, suggesting that hyaluronan-induced fast-proliferating PDMSCs may rely less on mitochondrial function as an energy source and induce a mitochondrial functional switch to glycolysis. Conclusions PDMSCs cultured on both CHA and SHA exhibited a reduction in reactive oxygen species levels. The results from this study clarify our understandings on the effect of hyaluronan on stem cells and provide important insights into the effect of distinct supplementation methods used during cell therapies.