The use of Methylphenidate (MP) can have adverse effects on bone growth and mineralization. This study aimed to investigate the underlying pathophysiology of MP-induced skeletal deficits in growing rats using stereological and immunohistochemical methods. Male rats, aged 4 weeks, were orally treated with MP through an 8-h/day water drinking protocol. The rats (n=30) were randomly divided into three groups: MP-High Dose (30/60 mg/kg/day MP), MP-Low Dose (4/10 mg/kg/day MP), and control (water only). After 13 weeks, the femoral bones were assessed using calliper measurements, dual-energy X-ray absorptiometry, and biomechanical evaluation. The total femur volume, cartilage volume, growth zone volume, and volume fractions were determined using the Cavalieri method. Immunohistochemical analyses were conducted using alkaline phosphatase and anti-calpain antibody staining. Rats exposed to MP exhibited significant reductions in weight gain, femoral growth, bone mineralization, and biomechanical integrity compared to the control group. The total femoral volume of MP-treated rats was significantly lower than that of the control group. The MP-High Dose group showed significantly higher ratios of total cartilage volume/total femoral volume and total growth zone volume/total femoral volume than the other groups. Immunohistochemical evaluation of the growth plate revealed reduced osteoblastic activity and decreased intracellular calcium deposition with chronic MP exposure. The possible mechanism of MP-induced skeletal growth retardation may involve the inhibition of intracellular calcium deposition in chondrocytes of the hypertrophic zone in the growth plate. In this way, MP may hinder the differentiation of cartilage tissue from bone tissue, resulting in reduced bone growth and mineralization.