Prohexadione-calcium (Pro-Ca), a plant growth regulator used primarily in fruit trees to suppress excessive vegetative growth and to inhibit disease incidence, is known to inhibit dioxygenase enzymes and to inhibit GA biosynthesis. It induces genes for polyphenols synthesis. The objective of this project was to determine if the bioregulator Pro-Ca would alter the yield of essential oil, secondary metabolites, and growth in spearmint. Spearmint shoot cuttings from the same mother plant were used in this study. The plants were treated with 0, 125, 250, 375, and 500 mg/L a.i. of Pro-Ca over four weeks, and growth responses were measured every week and at harvest. Compared with the untreated control plants, plant height, branch length, number of nodes, and fresh weight were decreased with increased concentration of Pro-Ca treatment, and total phenolics accumulation increased. Rosmarinic acid and total chlorophyll content were reduced relative to control after treatment. Treatment with increased concentration of Pro-Ca altered the accumulation of flavonoids compounds. Increased concentration of catechin and eriodictyol-7-glucoside, and decreased concentrations of procyanidin and luteolin occurred compared with the untreated plant. Modification of newly formed flavonoid synthesis could be used as a new potential strategy in plant protection.