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Simple Summary: Weaning is one of the biggest challenges in a pig's life. Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse stress conditions. There are two autophagy-modifying agents, rapamycin (RAPA) and chloroquine (CQ), that are often used in vitro and in vivo to regulate this process. We speculated that the regulation of autophagy may have some effect on weaning pressure. In this study, we try to understand the role of autophagy in intestinal barrier function and inflammation during the first week after weaning. We examined the effects of modulation of autophagy via RAPA and CQ on growth performance, immunity, inflammation profile, and the intestinal barrier to find potential value for CQ as a feed additive agent for ameliorating weaning stress. Early weaning stress impairs the development of gastrointestinal barrier function, causing immune system dysfunctions, reduction in feed intake, and growth retardation. Autophagy was hypothesized to be a key underlying cellular process in these dysfunctions. We conjectured that rapamycin (RAPA) and chloroquine (CQ), as two autophagy-modifying agents, regulate the autophagy process and may produce deleterious or beneficial effects on intestinal health and growth. To explore the effect of autophagy on early weaning stress in piglets, 18 early-weaned piglets were assigned to three treatments (each treatment of six piglets) and treated with an equal volume of RAPA, CQ, or saline. The degree of autophagy and serum concentrations of immunoglobulins and cytokines, as well as intestinal morphology and tight junction protein expression, were evaluated. Compared with the control treatment, RAPA-treated piglets exhibited activated autophagy and had decreased final body weight (BW) and average daily gain (ADG) (p < 0.05), impaired intestinal morphology and tight junction function, and higher inflammatory responses. The CQ-treated piglets showed higher final BW, ADG, jejuna and ileal villus height, and lower autophagy and inflammation, compared with control piglets (p < 0.05). Throughout the experiment, CQ treatment was beneficial to alleviate early weaning stress and intestinal and immune system dysfunction. [ABSTRACT FROM AUTHOR]