Dietary intake of vitamin D includes vitamin D3 (vitD3), 25-hydroxyvitamin D3 (25OH-D3), and vitamin D2 (vitD2). However, the bioactivity of the different species has not been scientifically established. The hypothesis in this study was that vitD3, 25OH-D3, and vitD2 have an equal effect on 25-hydroxyvitamin D in serum (vitamin D status). To test our hypothesis, we performed a randomized, crossover study. Twelve young males consumed 10 µg/day vitD3 during a four-week run-in period, followed by 3 × 6 weeks of 10 µg/day vitD3, 10 µg/day 25OH-D3, and 10 µg/day vitD2. The content of vitD3, vitD2, 25OH-D3, and 25-hydroxyvitamin D2 (25OH-D2) in serum was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The hypothesis that the three sources of vitamin D affect vitamin D status equally was rejected. Based on the assumption that 1 µg vitD3/day will show an increase in vitamin D status of 1.96 nmol/L, the results showed that 23 µg vitD2 and 6.8 µg 25OH-D3 was similar to 10 µg vitD3. These results demonstrate that further investigations are necessary to determine how to quantify the total vitamin D activity based on chemical quantification of the individual vitamin D metabolites to replace the total vitamin D activity assessed in biological rat models.