This study assessed the effect of probiotic supplementation on immune response in physically active males before and after exhausting exercise. With growing evidence linking gut health to immune function, the aim was to determine whether 12 weeks of probiotics could influence key immune markers in athletes. In this semi-experimental study, 28 male athletes (18–35 years) from sports centers in Osh City, Kyrgyzstan, who engaged in regular training (≥ 3 days/week for ≥ 3 years) were randomly assigned to probiotic or placebo groups. Baseline characteristics included age (20.98 ± 5.9 years), weight (62.3 ± 9.8 kg), height (168.2 ± 7.6 cm), BMI (22.05 ± 2.4 kg/m²), and VO₂ max (59.98 ± 8.8 ml/kg/min), with no significant differences between groups. Immune markers—cell blood count (CBC), C-reactive protein (CRP), and immunoglobulin A (IgA)—were monitored at rest and after exercise. Probiotic supplementation resulted in a significant increase in monocyte levels at rest and during activity compared to placebo. Within the probiotic group, lymphocyte, monocyte, and granulocyte percentages rose significantly during activity (p < 0.001). These results indicate that probiotics may positively modulate immune function in athletes under high physical stress. Given that intense training can suppress immunity and increase susceptibility to illness, probiotic use may enhance gut microbiota, supporting immune resilience. A balanced microbiome not only aids digestion and nutrient absorption but also plays a vital role in regulating inflammation and immune responses. These findings support probiotics as a potential strategy to maintain immune health in physically active populations, warranting further investigation into strain-specific effects and long-term benefits.