Gastrointestinal myofibroblasts are contractile, electrically nonexcitable, transitional cells that play a role in extracellular matrix production, in ulcer healing, and in pathophysiological conditions they contribute to chronic inflammation and tumor development. Na⁺/Ca²⁺exchangers (NCX) are known to have a crucial role in Ca²⁺homeostasis of contractile cells, however, no information is available concerning the role of NCX in the proliferation and migration of gastrointestinal myofibroblasts. In this study, our aim was to investigate the role of NCX in the Ca²⁺homeostasis, migration, and proliferation of human gastrointestinal myofibroblasts, focusing on human gastric myofibroblasts (HGMs). We used microfluorometric measurements to investigate the intracellular Ca²⁺and Na⁺concentrations, PCR analysis and immunostaining to show the presence of the NCX, patch clamp for measuring NCX activity, and proliferation and migration assays to investigate the functional role of the exchanger. We showed that 53.0 ± 8.1% of the HGMs present Ca²⁺oscillations, which depend on extracellular Ca²⁺and Na⁺, and can be inhibited by NCX inhibitors. NCX1, NCX2, and NCX3 were expressed at both mRNA and protein levels in HGMs, and they contribute to the intracellular Ca²⁺and Na⁺homeostasis as well, regardless of the oscillatory activity. NCX inhibitors significantly blocked the basal and insulin-like growth factor II-stimulated migration and proliferation rates of HGMs. In conclusion, we showed that NCX plays a pivotal role in regulating the Ca²⁺homeostasis, migration, and proliferation of HGMs. The inhibition of NCX activity may be a potential therapeutic target in hyperproliferative gastric diseases.