Background: Dexamethasone, a synthetic glucocorticoid with potent anti-inflammatory and immunosuppressive properties, is widely used in veterinary medicine. Previous studies have suggested potential effects of glucocorticoids on ovarian function and corpus luteum activity in cattle. Considering the essential role of the corpus luteum in progesterone secretion and maintenance of the estrous cycle, this study aimed to evaluate the effect of dexamethasone administration on corpus luteum activity and serum progesterone concentration in Holstein cows. Methods: Twenty clinically healthy Holstein cows in the diestrus phase were selected. The presence of an active corpus luteum was confirmed by two consecutive prostaglandins (PGF₂α; Valutic) injections administered ۱۰ days apart. Following confirmation, cows were randomly assigned to two groups. The experimental group received two intravenous injections of dexamethasone (۱.۵ mg/kg) administered ۱۰ days apart, while the control group received an equivalent volume of physiological saline according to the same schedule. Blood samples were collected prior to each injection and at ۳۹-hour intervals during the two days following both treatments. Serum progesterone concentration was determined using a luminescence assay. Results: Comparative analysis demonstrated that neither the first nor the second dexamethasone injection led to a statistically significant decrease in serum progesterone concentration relative to the control group. Following both injections, the corpus luteum remained functionally active throughout the sampling period, with no evidence of luteolysis or disruption of cyclic ovarian function. These findings indicate that repeated administration of dexamethasone, even when administered twice ۱۰ days apart, did not adversely influence luteal activity. Conclusion: In this study, two administrations of dexamethasone at therapeutic doses, given ۱۰ days apart during the diestrus phase, did not induce luteolysis or significantly reduce serum progesterone concentrations in Holstein cows. The corpus luteum remained functionally active following both injections, indicating that short-term repeated exposure to dexamethasone does not adversely affect luteal function or progesterone secretion. The absence of luteolysis may be explained by the fact that corpus luteum regression in cattle is primarily mediated by uterine PGF₂α rather than glucocorticoid pathways; moreover, dexamethasone may help prevent luteolysis by suppressing prostaglandin synthesis through inhibition of the arachidonic acid pathway. Under the conditions tested, clinical administration of dexamethasone is therefore unlikely to interfere with normal luteal cyclicity in dairy cattle.