The role of the antioxidant response system in association with the proline-associated pentose phosphate pathway for cold adaptation was investigated in three cool-season turfgrasses during a cold acclimation period. As phenolic biosynthesis and antioxidant stimulation is proposed to be linked to the proline-associated pentose phosphate pathway, this study was aimed to determine the active role of proline in metabolic regulation and its relationship with the cold stress tolerance mechanism of cool-season turfgrasses. In this study, significant accumulation of total soluble phenolics and higher total antioxidant activity was observed in creeping bentgrass (Agrostis stolonifera L.), kentucky bluegrass (Poa pratensis L.), and perennial ryegrass (Lolium perenne L.) during cold acclimation, confirming the direct and indirect role of phenolics to counter low temperature-induced oxidative stress. A positive correlation between high phenolic content and the proline-associated pentose phosphate pathway was also found in investigated turfgrass species during a cold acclimation period. Low succinate dehydrogenase activity along with the high glucose-6-phosphate dehydrogenase activity in cold-acclimated turfgrass species suggested a probable shift of carbon flux from the energy-consuming tricarboxylic cycle to the alternative energy-efficient proline-associated pentose phosphate pathway to induce a better cold stress tolerance mechanism in these cool-season turfgrasses. Higher proline accumulation in cold-acclimated turfgrass species also supported the above findings and a probable proline oxidation to support mitochondrial oxidative phosphorylation was observed in acclimated kentucky bluegrass based on the activity of proline dehydrogenase, which likely supports the active metabolic role of proline in stress-induced situations. Through this study, a significant variation in cold stress tolerance mechanisms was observed among three investigated cool-season turfgrass species during cold acclimation. Furthermore, a high cold stress tolerance characteristic was observed in kentucky bluegrass by adapting a more efficient pathway for an antioxidant response linked to proline accumulation.