<p>Cold stress severely affects cotton growth and yield worldwide. Mepiquat chloride (MC), a widely used plant growth regulator in cotton, has not been extensively studied under cold stress conditions. This study evaluated the effects of three MC levels (0 mM, 50 mM, and 100 mM) applied at 21 days after sowing (DAS) on the morpho-physiological and biochemical responses of two cotton varieties (Sikang 1 and Sikang 3) under normal temperature and cold stress during the seedling stage. The present study revealed that the MC application reduced leaf length, leaf width, shoot length, and plant height but increased total plant fresh weight and root growth attributes. Furthermore, the MC application enhanced chlorophyll pigments, protein content, superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) while reducing malondialdehyde (MDA). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses (representative images, <i>n</i> = 3 biological replicates) revealed that MC-treated plants exhibited more intact cell membranes, better organized chloroplasts, and preserved thylakoid structures under cold stress compared with untreated plants. The improvement in morpho-physiological activity was largely attributed to the increment in root growth characteristics. Overall, MC at 50 and 100 mM significantly strengthened root architecture, chlorophyll content, cellular integrity, and antioxidant defense, resulting in improved cold tolerance in cotton seedlings. Among the tested concentrations, 100 mM MC provided the most substantial protective effects, particularly in Sikang 1. The corresponding findings highlight MC as a promising regulator for alleviating cold stress in cotton, but its molecular and hormonal mechanisms require further investigation. </p> Graphical Abstract <p>Graphical representation of MC-mediated cold stress tolerance in cotton</p> <p></p>