Understanding the nature of drought stress in cultivated plants is perhaps the most notable challenge associated with global climate change, increasing the uncertainty associated with the expansion and productivity of major crops that respond to heat and water stress with reduced agronomic performance. In Colombia, the tendency has been to grow short-cycle horticultural species, such as butternut squash (Cucurbita moschata Duchesne), that present efficient water use response. To study the physiological response of three butternut squash cultivars (UNAPAL Abanico-75, UNAPAL Bolo Verde, UNAPAL Dorado) to soil moisture regime (Kc), four irrigation water depths in two consecutive trials (0.4; 0.6; 0.8 y 1.0 trial I y 0.6; 0.8; 1.0 y 1.2 trial II) were applied in a split-plot design with 8 replicates, totaling 96 plants, where the main plot was the irrigation treatment and subplots, the cultivars. Physiognomic variables (biomass, leaf area, number of stomata, root length) showed significant biological performance (P<0.05) when the maximum soil moisture regime (Kc=1.2) was applied. In contrast, plants presented significant discrepancies in dimension and appearance (P<0.05) in water deficit conditions (Kc=0.4), with a significant decrease (P<0.05) in leaf blade macromolecular composition (soluble protein, soluble carbohydrates, chlorophylls a and b, carotenes). Water deficit also triggered hydrogen peroxide (H2O2) production, to which plants responded by producing enzymes to protect photosynthesis reactions. Malondialdehyde, proline, superoxide dismutase, catalase, peroxidases, and ascorbate peroxidase therefore increased significantly (P<0.05), all of which help restrict H2O2 while protecting plant cells from lethal lipid peroxidation caused by free radicals, the latter being significant (P<0.05) in the tolerance response of cultivars Abanico-75 and Dorado to water deficit. Study results can serve as basis for future drought tolerance studies in squash.