Vitamin D dysregulation can occur in people living with HIV, disrupting calcium homeostasis, and bone turnover. We aimed to investigate the potential mechanisms by which vitamin D regulates bone turnover in adolescents living with perinatally-acquired HIV (ALWH) in Southern Africa. A pre-planned secondary analysis was performed of baseline data from the vitamin D for adolescents with HIV to reduce musculoskeletal morbidity and immunopathology trial (PACTR20200989766029) which enrolled ALWH (11-19 yr) taking antiretroviral therapy for ≥6 mo, and recorded socio-demographic, clinical and dietary data. After over-night fasting, vitamin D metabolites (25(OH)D, 1,25(OH)2D, and 24,25(OH)2D), intact parathyroid hormone (PTH), and bone turnover markers (BTMs) (C-terminal telopeptide of type I collagen (CTX) and procollagen type 1 N-terminal propeptide (P1NP)) were measured. Tandem Mass Spectrometry measured vitamin D metabolites, while intact PTH and BTMs were analyzed by electrochemiluminescence immunoassay. Stratified by 25(OH)D (<75 vs ≥75 nmol/L), associations between standardized concentrations (β = standard deviations) of vitamin D metabolites, intact PTH and BTMs were assessed using structural equations modelling (SEM) adjusted for age, sex, and country (Zimbabwe/Zambia). Among the 842 ALWH enrolled, the median dietary calcium intake was 100 mg (IQR: 55-145). The SEM showed PTH was positively associated (β: 0.21; 95% CI, 0.1, 0.32) with 1,25(OH)2D, only when 25(OH)D was <75 vs ≥75 nmol/L (β: 0.23; 95%CI, -0.13, 0.59), with evidence of an interaction (β: -0.11; 95%CI, -0.20, -0.02). A positive relationship between 25(OH)D and 24,25(OH)2D was seen irrespective of 25(OH)D concentration. 24,25(OH)2D was inversely related to BTMs, particularly when 25(OH)D was <75 nmol/L (CTX: β: -0.15; 95% CI, -0.24, -0.06 and P1NP: β: -0.14; 95%CI, -0.22, -0.06). There was interaction between dietary calcium and 25(OH)D on PTH (β: -0.15; 95% CI, -0.22, -0.07) suggesting an interaction between low 25(OH)D and low dietary calcium which increases PTH. In conclusion, associations between 25(OH)D, PTH, 1,25(OH)2D, and BTMs in ALWH appear dependent upon 25(OH)D concentrations <75 nmol/L and calcium intake. A novel, potentially causal pathway between 25(OH)D, 24,25(OH)2D, and BTMs was seen. Findings enhance understanding of vitamin D metabolism in people living with HIV.