Objective This study aims to investigate the impact of Tubeimoside I on the proliferation of liver cancer cells and its molecular mechanism. Methods MTS assay and colony formation assay were conducted to examine the effects of different concentrations of Tubeimoside I on the growth of MHCC97-H liver cancer cells. Glucose uptake, lactate production, LDH activity, extracellular pH, cellular oxygen consumption and mitochondrial respiratory chain complex IV activity were measured to analyze the influence of Tubeimoside I on glycolysis and oxidative phosphorylation in liver cancer cells. qRT-PCR and Western blot were performed to detect the expression of HIF1α in MHCC97-H liver cancer cells. Results Tubeimoside I treatment decreased the proliferation ability of liver cancer cells in a dose- and time-dependent manner. It also reduced glucose uptake, lactate production and LDH activity, increased extracellular pH, and enhanced cellular oxygen consumption and mitochondrial respiratory chain complex IV activity. Conclusion Tubeimoside I inhibits cell glycolysis and promotes oxidative phosphorylation by suppressing the expression of HIF-1α, thereby suppressing the proliferation of liver cancer cells