ClC-1 is the dominant sarcolemmal chloride channel and plays an important role in regulating membrane excitability that is underscored by ClC-1 mutations in congenital myotonia. Members of the ClC family have the unusual distinction of being divided into chloride channels and anion/H⁺ antiporters. Both subtypes share general architectural features, and all 9 mammalian ClC proteins have extensive intracellular carboxy-termini that contain tandem Cystathionine β-synthase (CBS) domains.Here we show that the co-enzyme β-nicotinamide adenine dinucleotide (NAD), an important metabolic regulator, robustly inhibits ClC-1 when included in the pipette solution in whole cell patch-clamp experiments, and when transiently applied to inside-out patches. The oxidized (NAD⁺) form of the co-enzyme was more efficacious than the reduced (NADH) form, and inhibition by both was greatly enhanced by acidification. Homology modelling, based on the crystallographic coordinates of the homologous ClC-5 and CmClC proteins, and in silico docking suggests that NAD⁺ binds with the adenine base deep in a cleft formed by ClC-1’s intracellular CBS domains and the nicotinamide base interacts with the membrane-embedded channel domain. Consistent with modelling predictions, mutation of residues in CBS and channel domains attenuated (G200R, T636A, H847A) or abrogated (L848A) the effect of NAD⁺. In addition, the myotonic mutations G200R and Y261C abolished potentiation of NAD⁺ inhibition at low pH. Our results identify a new biological role for NAD and suggest that the main physiological relevance of our findings may be the exquisite sensitivity to intracellular pH that NAD⁺ inhibition imparts to ClC-1 gating. Our findings are consistent with the reduction of sarcolemmal chloride conductance that accompanies acidification of skeletal muscle and suggest a previously unexplored mechanism in the pathophysiology of myotonia.