The increase in litter size has led to a higher proportion of low birthweight (LBW) piglets, which are disadvantaged in terms of later growth and organ maturation. Supplementing with glutamine (Gln) has been proposed to improve this situation. We investigated the metabolism of Gln and glucose (Glc) in LBW and normal (NBW) birthweight (BiW) suckling piglets supplemented with Gln or water (W). Forty-six neonatal male German Landrace piglets with a BiW of 1.04 ± 0.02 kg for LBW piglets and 1.55 ± 0.02 kg for NBW piglets were assigned to one of four treatments in a 2 × 2 factorial design (LBW/NBW; Gln/W). Half of the groups received either 1 g/kg bodyweight (BW)/d of Gln (LBW-Gln, NBW-Gln; n = 12) dissolved in water, or water (W) as a control (LBW-W, NBW-W; n = 11), until age 15 d. At 12 d, piglets were implanted a jugular vein catheter and then underwent oral tests: on 14 d with 13C5-Gln/Gln, on 15 d with lactulose/mannitol (Lac/Man) and on 16 d with 13C6-Glc/Glc and xylose (Xyl). After a baseline blood sample, blood samples were collected over 5 h. Enrichments of 13C tracers in plasma and 13CO2 in red blood cells (RBC) were measured, along with plasma concentrations of Lac, Man, Xyl, Glc, lactate, urea, and amino acids. We calculated plasma Gln and Glc rate of appearance (Ra), 13C recovery (13C REC) in RBC CO2 from 13C5-Gln and 13C6-Glc as a proxy of whole-body oxidation and 13C6-Gln conversion to 13C3-Glc to obtain a measure of gluconeogenesis. The BW, average daily gain (ADG), abdominal circumference (ABC), crown-rump length (CRL), body mass index (BMI), and ponderal index were measured. The BW of LBW piglets supplemented with Gln was lower than of those supplemented with W at 12, 13, and 15 d (P < 0.05). The BW, ADG, ABC, CRL, and BMI remained lower in LBW than in NBW piglets (P < 0.01). Plasma urea was higher in the Gln groups (P < 0.05). The Xyl and Man absorption tests indicated a greater intestinal absorptive capacity/area in LBW piglets (P < 0.05). The 13C REC values of Gln and Glc were higher in LBW than in NBW piglets (P < 0.01), whereas 13C REC of Gln was higher than that of Glc independently of BiW and supplementation (P = 0.005). Plasma 13C6-Glc Ra was higher than that of 13C5-Gln Ra in all groups (P < 0.01). The 13C5-Gln to 13C3-Glc conversion did not differ among groups (P > 0.1). In conclusion, LBW and NBW piglets differ in Gln and Glc metabolism, but Gln supplementation did not show a consistent effect on the measured parameters.