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Introduction: Vitamin B12 (VB12), particularly its active form methylcobalamin (MeB12), contributes to neuromuscular function and energy metabolism, which may be relevant for sports performance. However, evidence on the acute effects of MeB12 supplementation in athletes remains limited. Objective: To evaluate the effects of short-term (3-day) MeB12 supplementation on anaerobic and cognitive performance in amateur cyclists. Methods: A randomized, triple-blind, placebo-controlled crossover clinical trial was conducted in 18 amateur cyclists. Participants received formulations containing MeB12 (1 mg/day; MecobalActive®, HTBA) or placebo for three consecutive days. Anaerobic performance was assessed using a repeated Wingate protocol, and cognitive performance was evaluated using a light-based mental agility/reaction test system. Biochemical analyses included plasma VB12 concentrations. Primary outcomes included peak power output (absolute and relative), fatigue index across repeated sprints, and cognitive response time. Results: Compared with placebo, MeB12 supplementation was associated with higher peak power output, with increases in absolute maximal power (PMAX: +4.1%, p = 0.016) and relative maximal power (PMAXREL: +4.4%, p = 0.013). MeB12 supplementation was associated with a smaller decline in performance across repeated sprints, with a smaller drop in fatigue index from the first to the fifth sprint (p = 0.012). Pre-exercise cognitive performance improved, with a shorter total reaction test time (−4.9%, p < 0.001) versus placebo. Plasma VB12 concentrations increased by 16.8% following MeB12 supplementation. Conclusions: A brief, 3-day intervention with methylcobalamin (1 mg/day) was associated with positive changes, when compared with placebo, in selected markers of anaerobic performance (peak power and fatigue-related decline) and pre-exercise cognitive performance in recreationally trained amateur cyclists, suggesting a possible involvement of peripheral and central mechanisms.