Antibiotics have been used for over 60 years by the swine industry to improve growth performance and feed efficiency. With rising concerns over antimicrobial resistance and government restrictions such as the Veterinary Feed Directive on usage of in-feed antibiotics, alternatives to feeding antibiotic growth promoters (AGPs) to nursery pigs are needed. However, the mechanism of action by which AGPs work is poorly understood. Thus, the objective of this study was to investigate the mechanisms of action by which AGPs increase nursery pig performance. Over two replicates, 24 weaned pigs (6.75 ± 0.75 kg body weight) were randomly allotted to either control (CON, n = 12) or sub-therapeutic antibiotic (sCTC, n = 12) treatments and housed individually. A 2-phase corn-soybean-based nursery diet was fed, with the sCTC diets containing 40 ppm feed-grade chlortetracycline. Individual pig average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) were calculated weekly for 5 weeks. Thereafter, all pigs were euthanized and necropsied for tissue collection. The overall performance data indicated that sCTC pigs had increased ADG (0.43 vs. 0.32 kg/d, P = 0.001) and ADFI (0.51 vs. 0.37 kg/d, P = 0.002) compared with CON pigs; however, G:F was not different as a result of dietary treatment (0.85 vs. 0.88, P = 0.617). Intestinal barrier permeability, ileal active nutrient transport, and cecal short chain fatty acid concentrations did not differ (P > 0.10) due to dietary treatment, however changes in several ileum mRNA transcripts suggest that inflammation may be reduced in sCTC pigs. Further, the changes observed in the proteomes of the ileum, colon, skeletal muscle, and liver suggest that the sub-therapeutic mode of action of AGPs may include post-absorptive changes and warrants further investigation.