Mitochondria are a major source and target of endogenous Reactive Oxygen Species (ROS) that are produced as normal by-products of the electron transport system. Decay of mitochondrial function is a major contributor to aging process. The age-related overproduction of ROS compared to the scavenging efficiency of the antioxidant cellular system leads to the onset of an oxidative stress condition. The accumulation of oxidatively damaged macromolecules may alter the mitochondrial bioenergetic function and may play a key role in the functional decay of cells and tissues that is among the causes of aging and of the associated degenerative pathologies. Acetyl-L-carnitine (ALCAR) is a biomolecule able to limit age-linked mitochondrial decay in brain, liver, heart and skeletal muscles by increasing mitochondrial efficiency. Aim of this study was to test if long-term ALCAR supplementation to aged rats was able to activate the PGC-1alpha/PGC-1beta dependent mitochondrial biogenesis in liver tissue. Young (6-month old) and old rats (28-month old) were used. Old rats were daily supplemented with ALCAR for 1 and 2 months. MtDNA content was quantified by Real Time PCR and citrate synthase activity was determined as marker of mitochondrial mass content. The levels of PGC-1alpha, PGC-1beta, TFAM, NRF-1 as well as of ND1 (a mitochondrial DNA encoded subunit of mitochondrial respiratory chain) and of COX IV (a nuclear DNA encoded subunit of mitochondrial respiratory chain) were quantify. Furthermore, the levels of the antioxidant enzymes Peroxiredoxin III, exclusively located into mitochondria and involved in the regulation of mitochondrial H2O2 concentration, and of SOD2 as well as of Drp1 and of MFN2, two proteins implicated in the processes of mitochondrial fission and fusion, were also measured. The obtained results revealed that ALCAR administration to old rats is able to prevent in liver the age-dependent decrease of mitochondrial decay. In particular, ALCAR treatment was able to up regulate the PGC-1alpha/PGC-1beta dependent mitochondrial biogenesis activating pathway, and to increase the mtDNA content and the mitochondrial mass, reduced in the liver of old rats.