Vitamin D deficiency, which is usually detected by using immunoassays or the more reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) methods, has recently been considered a public health problem worldwide. However, the vitamin D status in Chinese populations, as measured using the LC-MS/MS method, is not available. The objective of this multicenter study was to determine the vitamin D status and prevalence of vitamin D deficiency by using a reliable method in 5 large cities in China. From May 1 to September 31, 2013, we conducted a multicenter study on 2173 apparently healthy adults who were recruited from 5 Chinese cities. The 25-hydroxyvitamin D 25OHD2 and 25OHD3 levels were measured using the LC-MS/MS method. Intact parathyroid hormone (iPTH), calcium, phosphorus, and alkaline phosphate levels were also measured using an automatic analyzer. The mean 25OHD level of all participants was 19.4 +/- 6.4 ng/mL (2.5-97.5%: 7.9-32.6 ng/mL), and only 109 (5.0%) participants had a 25OHD2 level >2.5 ng/mL (maximum, 22.4 ng/mL). In this study, the prevalence of severe vitamin D deficiency (<10 ng/mL), vitamin D deficiency (10-20 ng/mL), vitamin D insufficiency (20-30 ng/mL), and vitamin D sufficiency (>30 ng/mL) was 5.9%, 50.0%, 38.7%, and 5.4%, respectively. Women had a significant higher rate of deficiency than men (66.3% vs 45.3%, P<0.01). Participants aged 18 to 39 years had a lower 25OHD level than elderly individuals (>59 years). Lifestyle may influence the 25OHD level more than the latitude, with participants in Dalian having the highest 25OHD level and the lowest deficiency rate. The serum iPTH level showed a significant negative correlation with the 25OHD(2) level (r = -0.23, P<0.01) after correcting for age and sex. In conclusion, the present study evaluated the vitamin D status using a reliable method, and our results indicate that vitamin D deficiency is prevalent among all age groups in China, especially among younger adults. We also observed significant differences in the 25OHD levels according to sex, age, and region among apparently healthy individuals.