Vitamin D, an important constituent of human health, is produced through the exposure of human skin to short wave (280–315 nm) ultraviolet radiation (UV). We aimed to establish whether an urbanized environment with tall buildings in close proximity (an “urban canyon”) significantly reduced the capacity of sunlight to synthesize vitamin D, when compared with a typical suburban area (∼2.5 km away); and to investigate the association of UV and vitamin D production with pollution, temperature, and humidity. Measurements of ambient UV (295–400 nm) (using a portable photometer/radiometer and detector) and synthesized vitamin D (from an in vitro model) were taken regularly at urban and control sites over 3 months in Brisbane, Australia. During a typical 20 min measurement, urban and control sites received 0.26 and 1.03 W m−2 mean total UV respectively (P < 0.001), and produced 0.12 and 0.53 μg mL−1 mean vitamin D (P < 0.001). Pollution, temperature and humidity were not associated with UV or vitamin D production. This demonstrates a large difference in vitamin D synthesis between an urban canyon and a nearby control site. Although the results cannot be directly applied to humans, they emphasize the need for further study of human vitamin D production in urban environments.