© 2015 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim. The stability of DNA is highly dependent on the properties of the surrounding solvent, such as ionic strength, pH, and the presence of denaturants and osmolytes. Addition of pyridine is known to unfold DNA by replacing π-π stacking interactions between bases, stabilizing conformations in which the nucleotides are solvent exposed. We show here experimental and theoretical evidences that pyridine can change its role and in fact stabilize the DNA under acidic conditions. NMR spectroscopy and MD simulations demonstrate that the reversal in the denaturing role of pyridine is specific, and is related to its character as pseudo groove binder. The present study sheds light on the nature of DNA stability and on the relationship between DNA and solvent, with clear biotechnological implications. The thermal stability of duplex DNA can be decreased by addition of pyridine, a well-known denaturant. In acidic solutions, pyridine is protonated and displays its role as DNA renaturant, which increases the melting temperature of the duplex further than a solution of NaCl at the same concentration. This behavior is specifically observed for pyridine. ; Peer Reviewed