The content of vitamin D in pork produced in conventional systems depends on the vitamin D concentration in the pig feed. Both vitamin D3 and 25-hydroxyvitamin D3 (25(OH)D3) are essential sources of dietary vitamin D; however, bioavailability assessed by serum 25(OH)D3 concentration is reported to be different between the two sources. Furthermore, the relationship between serum 25(OH)D3 level and the tissue content of vitamin D3 and 25(OH)D3 is unknown. The objective of this study was to investigate the potential of increasing the content of vitamin D in different pig tissues by increasing the levels of vitamin D3 and 25(OH)D3 in the pig feed for 49 d before slaughter. Concurrently, the 25(OH)D3 level in serum was investigated as a biomarker to assess the content of vitamin D3 and 25(OH)D3 in pig tissues. Adipose tissue, white and red muscle, the liver and serum were sampled from pigs fed feed containing either vitamin D3 or 25(OH)D3 at 5, 20, 35 or 50 µg/kg feed for 7 weeks before slaughter. The tissue 25(OH)D3 level was significantly higher in the pigs fed 25(OH)D3 compared with those fed vitamin D3, while the tissue vitamin D3 level was higher in the pigs fed vitamin D3 compared with those fed 25(OH)D3. The content of 25(OH)D3 in the different tissues fully correlated with the serum 25(OH)D3 level, whereas the correlation between the tissue content of vitamin D3 and serum 25(OH)D3 was dependent on the source of the ingested vitamin D3.