The present project aimed to estimate bioavailability of dietary vitamin B12, for which little information is available in growing pigs. Two approaches, each using 2 quantities of dietary cyanocobalamin, were compared; the first was based on whole body retention for 8 d and the second was based on nycthemeral portal net flux of vitamin B12. In the first trial, 15 blocks of 3 pigs (31.7 ± 0.5 kg of BW) were formed according to their vitamin B12 status. Within each block, 1 pig (CONT) was killed and tissues were sampled for vitamin B12 determination. The remaining 2 piglets were fed 25 (B12-25) or 250 (B12-250) μg daily of cyanocobalamin for 8 d. Urine was sampled twice daily, and the pigs were killed and sampled as CONT pigs. The total content of vitamin B12 in the carcass, urine, and intestinal tract was affected by the dietary treatments (P < 0.01) but not in the liver (P > 0.019). The whole body retention of vitamin B12 was greater (P = 0.02) in B12-250 than B12-25 pigs, but the corresponding bioavailability was estimated to be 5.3 and 38.2%, respectively. In trial 2, 11 pigs (35.1 ± 4.0 kg of BW and 75.4 ± 5.9 d of age) fed a diet unsupplemented with vitamin B12 from weaning at 28 d of age were surgically equipped with catheters in the portal vein and carotid artery and an ultrasonic flow probe around the portal vein. Each pig received 3 boluses of 0 (B12-0), 25, and 250 μg of dietary vitamin B12 according to a crossover design. Postprandial nycthemeral arterial plasma concentrations of vitamin B12 reached minimum values (P < 0.01) between 15 and 18 h postmeal that were 29.6, 15.6, and 10.0% less than the premeal values for B12-0, B12-25, and B12-250 pigs, respectively (linear, P < 0.01). The cumulative net flux of vitamin B12 for 24 h corresponded to 2.4 and 5.1 µg for B12-25 and B12-250 treatments, respectively, and the corresponding bioavailability was estimated to be 9.7 and 2.0%, respectively. Although bioavailability estimates varied according to approaches, both showed the inverse relationship between dietary vitamin B12 and bioavailability of the vitamin. The dietary supplement of 25 µg was sufficient to maximize hepatic vitamin B12 retention and to attenuate the nycthemeral decrease of arterial plasma concentration of the vitamin.