High-intensity physical exercise favors anaerobic glycolysis and increases lactatemia. Lactate is converted back to glucose in the liver, so that the lactate threshold, an indicator of physical performance, must be related to the gluconeogenic capacity of the liver. This research assessed the effect of a high-intensity interval resistance training (HIIRT) on liver gluconeogenesis from lactate. Swiss mice were trained (groups T) on vertical ladder with overload of 90% of their maximal load. Control animals remained untrained (groups C0 and C8). In situ liver perfusion with lactate and adrenaline was performed in rested mice after six hours of food deprivation. There were larger outputs of glucose (T6 71.90%, T8 54.53%) and pyruvate (T8 129.28%) (representative values for 4 mM lactate) in the groups trained for six or eight weeks (T6 and T8), and of glucose in the presence of adrenaline in group T8 (280%). The content of PEPCK, an important regulatory enzyme of the gluconeogenic pathway, was 69.13% higher in group T8 than in the age-matched untrained animals (C8). HIIRT augmented liver gluconeogenesis from lactate and this might improve the lactate threshold. Bullet points: The liver metabolizes lactate from muscle into glucose. Physical training may enhance the gluconeogenic capacity of the liver. As lactate clearance by the liver improves, lactate threshold is displaced to higher exercise intensities.