S100A1, a 21-kDa dimeric Ca2+-binding protein of the EF-hand type, is expressed in cardiomyocytes and is an important regulator of heart function. During ischemia, cardiomyocytes secrete S100A1 to the extracellular space. Although the effects of extracellular S100A1 have been documented in cardiomyocytes, it is unclear whether S100A1 exerts modulatory effects on other tissues in proximity with cardiac cells. Therefore, we sought to investigate the effects of exogenous S100A1 on Ca2+ signals and electrical properties of superior cervical ganglion (SCG) neurons. Immunostaining and Western blot assays indicated no endogenous S100A1 in SCG neurons. Cultured SCG neurons took up S100A1 when it was present in the extracellular medium. Inside the cell exogenous S100A1 localized in a punctate pattern throughout the cytoplasm but was excluded from the nuclei. S100A1 partially colocalized with markers for both receptor- and non-receptor-mediated endocytosis, indicating that in SCG neurons multiple endocytotic pathways are involved in S100A1 internalization. In compartmentalized SCG cultures, axonal projections were capable of uptake and transport of S100A1 toward the neuronal somas. Exogenous S100A1 applied either extra- or intracellularly enhanced Cav1 channel currents in a PKA-dependent manner, prolonged action potentials, and amplified action potential-induced Ca2+ transients. NMR chemical shift perturbation of Ca2+-S100A1 in the presence of a peptide from the regulatory subunit of PKA verifies that S100A1 directly interacts with PKA, and that this interaction likely occurs in the hydrophobic binding pocket of Ca2+-S100A1. Our results suggest the hypothesis that in sympathetic neurons exogenous S100A1 may lead to an increase of sympathetic output.