Acute exercise is known to influence several biochemical parameters, including plasma homocysteine (Hcy), a sulfur-containing amino acid linked to cardiovascular risk. The metabolism of Hcy is dependent on several B-vitamins, notably vitamin B2 (riboflavin), B12 (cobalamin), and folate (vitamin B9), which act as coenzymes or methyl donors in one-carbon metabolic pathways. This study examined how a single session of moderate-to-intense aerobic exercise alters the levels of these vitamins and whether these changes are associated with shifts in plasma homocysteine. Twenty healthy young adults performed a 45-minute treadmill exercise at 75% of maximum heart rate. Blood samples were collected at baseline, immediately post-exercise, and one hour post-exercise. The findings revealed a significant increase in homocysteine levels immediately post-exercise, accompanied by reductions in serum vitamin B2 and folate. No significant change was observed in vitamin B12. These findings suggest that acute exercise induces short-term disruptions in B-vitamin status that may contribute to transient elevations in homocysteine, underlining the need for monitoring nutritional status in physically active individuals.