A pilot study was performed to investigate the impact of dietary vitamin D on circulating 25-hydroxyvitamin D (25[OH]D) metabolite concentrations in sugar gliders (Petaurus breviceps). The study with diets containing 0, 0.2 (low), or 0.4 (moderate) International Units vitamin D3 per gram of dry matter and fed to adults at 2 locations. Serum 25[OH]D concentrations did not differ between animals fed produce only (no added vitamin D–either D2 or D3) for 3 weeks (8.83 ± 0.98 nmol/L), n = 6, or low dietary levels (7.86 ± 3.80 nmol/L), n = 7, continuously for multiple years. Conversely, animals consuming diets containing moderate vitamin D3 levels displayed increased circulating concentrations (15.00 ± 3.59), n = 8, after 3 weeks. Despite the response to diets supplemented with vitamin D, overall metabolite levels were low and may indicate minimal metabolic dependence on this nutrient in sugar gliders, similar to processes documented in other hindgut fermenters.