The aqueous environment and ionic surroundings are the most important factors that determine the conformation of DNA and its functioning in the cell. The specificity of the interaction between DNA and cations is especially pronounced with a decrease in the activity of water. In this work, we use FTIR spectroscopy to study the B–A transition in high-molecular-weight DNA with a decrease in the humidity of the film with different contents of Na ⁺ ions. The IR spectrum of DNA is very sensitive to its secondary structure. As well, this makes it possible to evaluate the amount of DNA-bound water. Dehydration of the DNA film leads to changes in the IR absorption spectrum characteristic of the B–A transition. Using thermogravimetric analysis, we have shown that the degree of DNA hydration reaches the saturation level at a relative humidity of 60% and decreases slightly upon further drying. The amount of water that is strongly bound to DNA has been found to decrease with increasing Na ⁺ concentration. In this case, sodium ions destroy the hydration shell of DNA and can interact directly with phosphate groups.