Weaning is a complex stressor associated with social, environmental and metabolic stress in pigs, that can be compounded by the negative effects of transport and thermal stress. To combat these negative effects, dietary antibiotics may be provided to newly weaned and transported piglets to reduce pathogen load and promote growth. However, their use may be limited in the future due to concerns regarding antibiotic resistance, thereby putting the health and welfare of piglets at risk and reducing overall productivity. We determined that withholding dietary antibiotics after weaning and simulated transport had a negative effect on piglet welfare; however, heat stress during simulated transport did not significantly impact post-transport welfare. Specifically, withholding dietary antibiotics increased intestinal damage, reduced FI and growth, and increased behaviors associated with illness in pigs (i.e., lying behavior) compared to piglets provided either dietary antibiotics or L-glutamine at 0.20% as-fed. These data provide further evidence that eliminating dietary antibiotics after weaning and transport will negatively impact the welfare and health of piglets, and may also indicate that the inclusion of L-glutamine could be a viable alternative to dietary antibiotics in production systems. However, because piglets were weaned and transported under simulated conditions, any implications towards the use of L-glutamine as an antibiotic alternative would need to be confirmed under commercial production conditions.