A randomized complete block design experiment was conducted to determine the safety and efficacy of supplementation of increasing concentrations of a novel, bacterial fermentation-derived vitamin D source on growth performance and tissue deposition of 25-hydroxycholecalciferol (25OHD3) in growing swine. Dietary treatments were as follows: commercial control with vitamin D3 (CON) at NRC recommended concentrations and 3 diets composed of CON + increasing inclusions (25, 50, and 250 µg/kg equivalent) of 25OHD3 from a novel source (CON+25; CON+50; and CON+250 respectively). Pigs (n = 144) were assigned to 24 pens which were allotted to 1 of the 4 dietary treatments and fed for 42 d. Blood samples were collected for 25OHD3 concentration determination and individual body weights were measured on experimental d 0, 39, and 63. On d 42, tissues from 48 pigs (12 pigs per dietary treatment) were analyzed for 25OHD3 concentration. No differences were observed in growth performance. Day 39 serum 25OHD3 concentrations were greatest in CON+250-fed pigs and linearly decreased as dietary 25OHD3 inclusion decreased (P < 0.0001). On d 42, tissue 25OHD3 concentrations increased linearly as 25OHD3 increased in the diet (P < 0.0001). On d 63, 21 d after dietary 25OHD3 withdrawal, serum 25OHD3 concentrations of all 25OHD3-fed pigs decreased to that of or within 2.76 ± 0.89 ng/mL of CON-fed pigs which demonstrates that feeding 250 µg/kg 25OHD3 is well tolerated by growing pigs and will clear the body within 21 d.