Glycine betaine is the main osmolyte synthesized and accumulated in mammalian renal cells. Glycine betaine synthesis is catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH) using NAD + as the coenzyme. Previous studies have shown that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD + with different affinities at each active site and that the binding is K + dependent. The objective of this work was to analyze the changes in the pkBADH secondary and tertiary structure resulting from variable concentrations of NAD + and the role played by K + . Intrinsic fluorescence studies were carried out at fixed-variable concentrations of K + and titrating the enzyme with varying concentrations of NAD + . Fluorescence analysis showed a shift of the maximum emission towards red as the concentration of K + was increased. Changes in the exposure of tryptophan located near the NAD + binding site were found when the enzyme was titrated with NAD + in the presence of potassium. Fluorescence data analysis showed that the K + presence promoted static quenching that facilitated the pkBADH-NAD + complex formation. DC data analysis showed that binding of K + to the enzyme caused changes in the α-helix content of 4% and 12% in the presence of 25 mM and 100 mM K + , respectively. The presence of K + during NAD + binding to pkBADH increased the thermal stability of the complex. These results indicated that K + facilitated the pkBADH-NAD + complex formation and suggested that K + caused small changes in secondary and tertiary structures that could influence the active site conformation.