Studies have shown that natural products can induce paraptosis in tumour cell lines. Paraptosis is characterized by cytoplasmic vacuolation arising from the endoplasmic reticulum (ER) and mitochondria. The mechanism of paraptosis is unclear; however, dysregulation of Ca homeostasis is believed to affect paraptosis induction. This study investigated the mechanism of cell death induced by a phytocannabinoid ratio in the MCF7 breast cancer cell line. The crystal violet assay was used to detect changes in viability and morphology changes were investigated using light and transmission electron microscopy. Various inhibitors, fluorescent staining with high-content screening, and Western blot analysis were used to investigate different cell death mechanisms. The phytocannabinoid ratio induced significant cell death and cytoplasmic vacuolation in MCF7 cells; however, no apoptosis, necrosis, autophagy, or ferroptosis was detected. Vacuolation induced by phytocannabinoid treatment was inhibited by cycloheximide, suggesting paraptosis induction. The mechanism of paraptosis induction was investigated, and it was found that treatment (1) induced ER dilation and mitochondrial swelling, (2) induced significant ER stress and mitochondrial Ca overload and dysfunction, which appeared to be mediated by the voltage-dependent anion channel, and (3) significantly impaired all mitochondrial metabolic pathways. The data demonstrated that paraptosis induced by the cannabinoid ratio was mediated by Ca flux from the ER to the mitochondria. These findings highlight a novel mechanism of cannabinoid-induced cell death and emphasize the anti-cancer potential of cannabinoid ratios, which exhibited enhanced effects compared to individual cannabinoids.