Skin cancer is the most common group of cancers globally, and solar ultraviolet (UV) radiation is the main cause. However, exposure to UV radiation also triggers vitamin D synthesis, crucial for bone health and immune function. While sun protection is essential for skin cancer prevention, the effect of high-SPF (sun protection factor) sunscreen on vitamin D levels is unclear. The Sun-D Trial sought to investigate the impact of routine application of broad-spectrum SPF 50+ sunscreen on serum 25-hydroxyvitamin D [25(OH)D] concentration and to determine whether any effects varied among subgroups defined by UV radiation at place of residence, baseline 25(OH)D concentration, and time outdoors. We randomised 639 Australians to daily sunscreen application or control (discretionary sunscreen use); serum 25(OH)D concentration was measured at baseline, 6 and 12 months. The main outcome was the change in serum 25(OH)D concentration. Of those recruited, 628 (98%) provided at least one follow-up blood sample. Their median age was 53 years, 66% were female, and 55% had a university qualification. The mean 25(OH)D concentration in both groups combined was 63 nmol/L at baseline (winter), 77.7 nmol/L at the end of summer, and 57.6 nmol/L at the final follow-up (winter). Findings from the Sun-D trial will inform strategies for optimising sun protection while maintaining healthy vitamin D status.