Little information is available on the effects of intensive greenhouse cultivation on selenium (Se) content in vegetables. This study was conducted in a typical greenhouse vegetable cultivation field in Nanjing. Soil samples as well as corresponding vegetables were collected, and Se contents were analyzed to reveal the factors affecting vegetable Se content. The health risk of Se intake through vegetables was also evaluated. The results showed that the contents of Se in vegetables varied with vegetable species and followed the order: leafy vegetable > tuber vegetable > fruit vegetable. Vegetable Se content decreased with increased soil organic matter (OM) content and organic bound Se content. This might be due to the large amounts of organic fertilizer input, which increased soil OM content as well as organic bound Se content, and thus decreased soil Se availability. The background soil Se level in study area was low. The decrease in vegetable Se content induced by application of organic fertilizer increased the health risk of Se deficiency to human.