The objective of this study was to evaluate the effects of dietary addition of lactic acid and glutamine, and their interactions, on growth performance, nutrient digestibility, digestive enzyme activity, intestinal barrier functions, microflora, and expressions of intestinal development-related genes of weaning piglets. Ninety-six 24-day-old weaning piglets (Duroc × Landrace × Yorkshire, weaned at 21 ± 1 d and fed the basal diet for a 3 d adaptation period) with initial body weight of 7.24 ± 0.09 kg were randomly assigned to one of four dietary treatments with six replicates per treatment and four pigs per replicate in a 2 × 2 factorial treatment arrangements: (1) CON (a 2-period basal diet; control), (2) LS (supplemented with 2% lactic acid), (3) GS (supplemented with 1% glutamine), and (4) LGS (supplemented with 2% lactic acid and 1% glutamine). The study lasted for 28 d. On days 25–28, fresh fecal samples were collected to evaluate apparent total tract digestibility (ATTD) of nutrients. After 28 d, one weaning pig per pen was euthanized, and physiological samples obtained. Results showed that the supplementation of lactic acid improved the ADFI of the pigs (p < 0.05), while the pigs fed the glutamine diet had a greater ADFI and higher G/F (p < 0.05), and there were significant interactive effects between lactic acid and glutamine on the ADFI and G/F of the pigs (p < 0.05). The ATTD of CP and ash for pigs fed with lactic acid was significantly enhanced, and pigs fed the glutamine diet had greater ATTD of CP and ash (p < 0.05), while there were significant interactive effects between lactic acid and glutamine on the ATTD of CP and ash of the pigs (p < 0.05). Pigs fed with lactic acid exhibited greater activity of α-amylase and lipase (p < 0.05); moreover, the activity of lipase in the pigs showed a significant interactive effect between lactic acid and glutamine (p < 0.05). There was a greater villus height and villus height to crypt depth ratio in pigs fed with lactic acid (p < 0.05), and the villus height to crypt depth ratio of pigs fed with glutamine was greater (p < 0.05). There were greater GLUT2, IGF-1, TGF-β2, OCLN, and ZO-1 mRNA levels in pigs fed with lactic acid (p < 0.05), and the supplementation of glutamine increased SGLT1, GLUT2, PepT1, IGF-1, IGF-1R, TGFβ-2, GLP-2, and OCLN mRNA levels (p < 0.05), Additionally, expressions of SGLT1, GLUT2, PepT1, IGF-1, IGF-1R, TGFβ-2, GLP-2, CLDN-2, OCLN, and ZO-1 mRNA levels of pigs showed a positive interactive effect between lactic acid and glutamine (p < 0.05). Supplementation of lactic acid significantly increased the populations of Bifidobacterium in cecal digesta, Lactobacillus in colonic digesta, and the content of butyric acid in colonic digesta (p < 0.05). In addition, there were significant interactive effects between lactic acid and glutamine on populations of Bifidobacterium in cecal digesta, Lactobacillus in colonic digesta, and the content of acetic acid, butyric acid, and total VFAs in cecal digesta of the pigs (p < 0.05). Collectively, the current results indicate that dietary supplementation with lactic acid and glutamine had a positive synergistic effect on weaning pigs, which could improve growth performance through promoting the development of the small intestine, increasing digestive and barrier function, and regulating the balance of microflora in pigs, and which might be a potential feeding additive ensemble to enhance the health and growth of weaning piglets in the post-antibiotic era.