The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria. ; We thank Gilles Mirambeau, Dmitry Temiakov and Arka Chakraborty for valuable discussion. The Structural Biology Unit is “Maria de Maeztu” Unit of Excellence awarded by Ministry of Economy and Competitiveness (MINECO) [MDM-2014-0435]. This study was supported by MINECO (BFU2009-07134, BFU2012-33516/ BMC, BFU2015-70645-R), Generalitat de Catalunya (SGR2009-1366, SGR2014-997), the European Union (FP7-HEALTH-2010-261460, FP7-PEOPLE-2011-290246, FP7-HEALTH-2012-306029-2) and the Spanish Research Council for a JAE fellowship to ARC. ; Peer reviewed