Betaine is an osmolyte with the potential to increase volatile fatty acids (VFAs) production and hence improve intestinal health.The present study investigated how betaine affects portal and arterial concentrations and net portal absorption (NPA) of VFA in growing Iberian pigs. Eight 30 kg BW Iberian growing barrows with indwelling catheters in portal vein, ileal vein and carotid artery were randomly assigned to a control diet or a diet supplemented with 0.5% betaine. Para-aminohippuric acid was infused into the ileal vein as a marker to determine portal blood flow using the dilution method. Blood samples were simultaneously taken from the carotid artery and portal vein at -60, 60, 120, 180, 240, 300 and 360 min after feeding 1 200 g of the diet. The NPA of VFA (acetate, propionate, butyrate, valerate, isobutyrate and caproate) was determined by multiplying the porto-arterial plasma concentration differences by portal plasma flow. Betaine increased NPA of acetate (1.44 fold; P < 0.001) and total VFA (0.55 fold; P < 0.001) while decreased NPA of propionate (-0.38 fold; P < 0.05) and valerate (-1.46 fold; P < 0.05) compared with control pigs. Estimated heat production potentially derived from NPA of VFA accounted for 0.20-0.27 of metabolizable energy for maintenance. Acetate and propionate accounted for most of the total VFA estimated heat production (0.83-0.89). Regarding bacterial communities, betaine apparently did not change the DNA abundance of fecal total bacteria, Lactobacillus, Bifidobacterium, Enterobacteriaceae, Bacteroides and the Clostridium clusters I, IV and XIV. In conclusion, betaine increased portal appearance and NPA of VFA, contributing to cover maintenance energy requirements.