DNAzymes have become attractive due to their potential biomedical and biotechnological applications, as well as their advantages in terms of stability, efficiency and synthetic accessibility with respect to protein or RNA catalysts. However, a lack of knowledge about the catalytic mechanisms of DNAzymes hampers further developments. Here, by means of high-level quantum mechanics/molecular mechanics simulations and biochemical studies, we determine the mechanism of RNA ligation catalysed by the 9DB1 DNAzyme. Our findings show that the mechanism consists of a single concerted asynchronous transition state where the O3′ atom of the acceptor RNA first attacks the α-phosphate group of the donor nucleotide, whereas the leftover proton from the O3′ atom is then transferred to the DNA. The mechanism involves the active participation of two Mg2+ ions, not present in the crystal structure but for which clear binding sites can be located.