Extremely low-frequency electromagnetic field (ELF-EMF) exposures influence many biological systems. These effects are mainly related to the intensity, duration, frequency, and pattern of the ELF-EMF. Our intent was to characterize the effect of specific pulsed electromagnetic fields on the in vitro proliferation of MCF-7 adenocarcinoma and MDA-MB-231 breast cancer cell lines and one non-cancerous M10 breast epithelial cell line. The following four important parameters of ELF-EMF were examined: frequencies (7.83 ± 0.3, 23.49 ± 0.3, and 39.15 ± 0.3 Hz), flux density (0.5 and 1 mT), exposure duration (12, 24, and 48 h), and the exposure methodology (continuous exposure versus switching exposure). The viability of MDA-MB-231 cells exposed to the optimized ELF-EMF pattern (7.83 ± 0.3 Hz, 1 mT, and 6 h switching exposure) was 40.1%. By contrast, the optimized ELF-EMF parameters that were most cytotoxic to breast cancer MDA-MB-231 cells were not damaging to normal M10 cells. In vitro studies also showed that exposure of MDA-MB-231 cells to the optimized ELF-EMF pattern promoted Ca influx and resulted in apoptosis. These data confirm that exposure to this specific ELF-EMF pattern can influence cellular processes and inhibit cancer cell growth. The specific ELF-EMF pattern determined in this study may provide a potential anti-cancer treatment in the future.