Background Voltage-sensitive dyes are important tools for mapping electrical activity in the heart. However, little is known about the effects of voltage-sensitive dyes on cardiac electrophysiology. Objective To test the hypothesis that the voltage-sensitive dye di-4-ANEPPS modulates cardiac impulse propagation. Methods Electrical and optical mapping experiments were performed in isolated Langendorff perfused guinea pig hearts. The effect of di-4-ANEPPS on conduction velocity and anisotropy of propagation was quantified. HeLa cells expressing connexin 43 were used to evaluate the effect of di-4-ANEPPS on gap junctional conductance. Results In electrical mapping experiments, di-4-ANEPPS (7.5 μM) was found to decrease both longitudinal and transverse conduction velocities significantly compared with control. No change in the anisotropy of propagation was observed. Similar results were obtained in optical mapping experiments. In these experiments, the effect of di-4-ANEPPS was dose dependent. di-4-ANEPPS had no detectable effect on connexin 43–mediated gap junctional conductance in transfected HeLa cells. Conclusion Our results demonstrate that the voltage-sensitive dye di-4-ANEPPS directly and dose-dependently modulates cardiac impulse propagation. The effect is not likely mediated by connexin 43 inhibition. Our results highlight an important caveat that should be taken into account when interpreting data obtained using di-4-ANEPPS in cardiac preparations.