Abstract This study tested the hypothesis that dietary supplementation with glycine (Gly) enhances the synthesis and availability of creatine (Cr) in tissues of pigs with intrauterine growth restriction (IUGR). At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of two groups, namely, supplementation with 1% Gly or 1.19% L-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Blood, kidneys, liver, pancreas, jejunum, longissimus lumborum muscle (LLM), and gastrocnemius muscle (GM) were obtained from the pigs within 1 wk after the feeding trial ended at 188 d of age to determine concentrations of guanidinoacetate (GAA), Cr, creatinine, and phosphocreatine (CrP). The organs were also analyzed for activities and mRNA levels for Cr-synthetic enzymes: L-arginine:glycine amidinotransferase (AGAT; forming GAA from Gly and L-arginine) and guanidinoacetate N-methyltransferase (GAMT; converting GAA and L-methionine into Cr). AGAT activity was present in the kidneys, liver, and pancreas, whereas GAMT activity was found in all the organs analyzed. AGAT and GAMT were most active per g of tissue in the kidneys and liver, respectively. Based on tissue mass, the kidneys had the greatest (P < 0.001) AGAT activity per whole organ, followed by the liver, while skeletal muscle had the greatest (P < 0.001) GAMT activity per whole organ, followed by the liver. Thus, the kidneys played a dominant role in forming GAA, whereas skeletal muscle and liver were the major sites for converting GAA into Cr. Dietary supplementation with 1% Gly enhanced AGAT activity in the kidneys and pancreas but reduced GAMT activity in the pancreas and small intestine, therefore directing GAA to the liver and skeletal muscle for Cr production. IUGR selectively reduced the concentration of Cr in LLM among all the organs studied. Except for the GM that had greater mRNA levels for GAMT in IUGR pigs, neither Gly nor IUGR affected mRNA levels for the selected genes in the tissues examined. Collectively, these novel results indicate that dietary Gly intake upregulates the Cr-synthetic pathway in swine.