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Background: Physical exercise activates the hypothalamopituitary-adrenal (HPA) axis and induces the body's inflammatory response. Due to contemporary dietary habits and increased energy expenditure, athletes are susceptible to depletion of magnesium ions. The aim of our study was to investigate, through assessment of plasma ACTH, serum IL-6, and salivary/serum cortisol levels, if chronic magnesium supplementation might reduce damaging stress effects in amateur rugby players. Methods: Rugby players (N = 23) were randomly assigned to intervention and control group. Basal samples were collected before intervention group started a 4-week-long supplementation with magnesium (500 mg Mg/d). Blood and saliva sampling were done a day before the match (Day-1), on the morning of competition (Game), and during a six-day-long recovery period (Day1, Day3 and Day6). ACTH, serum/salivary cortisol, IL-6 and total/differential leukocytes counts were determined at each time point. Results: There was a statistically significant increase in ACTH concentration in intervention group compared to control group, while reductions in cortisol concentrations between the two groups were the greatest at Day-1 (p< 0.01) and at the day of competition (Game) (p< 0.01). Our results revealed that magnesium completely abolished the increase in IL-6 level noted in control group on Day1 and Day3 vs. Day-1 (p< 0.01) and also diminished the rise in neutrophil/lymphocyte ratio in intervention group vs. control group (p< 0.01). Conclusions: These results suggest the possibly important influence magnesium supplementation might have on the change of parameters of HPA axis activity and reduction of immune response activation following strenuous physical exercise such as a rugby game.