Overtraining syndrome is a condition resulting from excessive training load associated with inadequate recovery and poor sleep quality, leading to performance decrements and fatigue. Here we hypothesized that vitamin D (VitD) deficiency is a lead factor in the development of the overtraining syndrome. To test this hypothesis, two groups of 60-week-old C57BL/6 mice followed a 16-week excessive eccentric-based overtraining by excessive downhill running with or without dietary VitD depletion (EX and EX-D- groups). Two control groups were trained by uphill running at the same load with or without VitD depletion (CX and CX-D- groups). Handgrip strength decreased throughout the protocol for all groups but the decrease was sharper in EX-D- group (VitD x training, p = 0.0427). At the end of the protocol, the mass of Triceps brachii muscle, which is heavily stressed by eccentric contractions, was reduced in eccentric-trained groups (training effect, p = 0.0107). This atrophy was associated with a lower concentration of the anabolic myokine IL-15 (training effect, p = 0.0314) and a tendency to a higher expression of the atrogene cathepsin-L (training effect, p = 0.0628). VitD depletion led to a 50% decrease of the fractional protein synthesis rate in this muscle (VitD effect, p = 0.0004) as well as decreased FGF21 (VitD effect, p = 0.0351) and increased osteocrin (VitD effect, p = 0.038) concentrations that would lead to metabolic defects. Moreover, the proportion of anti-inflammatory Th2 lymphocytes was significantly decreased by the combination of eccentric training with VitD depletion (vitD x training, p = 0.0249) suggesting a systemic inflammation. Finally, exploratory behavior time of mice was decreased by VitD depletion (VitD effect, p = 0.0146) suggesting a cognitive dysfunction. Our results suggest that VitD deficiency exacerbates the effects of overtraining.