Research on stress damage induced by weaning and its underlying mechanisms in squabs is notably scarce. The study was designed to uncover the potential mechanisms behind the intestinal epithelial barrier impairment due to early weaning (EW) in squabs by evaluating the function of intestinal epithelial barrier, the balance of T helper cell (Th) subsets, and the link between them. A total of 160 hatched squabs were randomly assigned to two groups: one received artificial pigeon milk starting from day 7 post-hatching, while the other group continued to be nourished by their parent pigeons. Ileal tissue and serum samples from eight replicates were gathered for analyses at intervals of 1, 4, 7, 10, 14, and 21 days after weaning. Results showed that body weight of squabs in the EW group decreased significantly from 1 day after weaning and continued throughout the experiment period. The serum endotoxin, diamine oxidase of weaned squabs increased significantly. The mRNA expression of ileal tight junction proteins of weaned squabs was significantly downregulated at multiple time points from 1 to 21 days after weaning. Compared to squabs in the control group, the weaned squabs exhibited immune imbalances of Th1/Th2 and Th17/Treg in ileum, characterized by abnormal expression of specific transcription factors of Th1, Th2, Th17, and Treg, as well as abnormal concentrations of differentiation-inducing cytokines and effector cytokines. Mantel tests showed that the changes of factors related to the differentiation of Th17/Treg cell subsets were significantly correlated with the diamine oxidase, endotoxin level, and the CDLN1 mRNA expression. Summarily, EW could lead to impaired growth, compromised intestinal epithelial barrier function and an imbalance in the differentiation of Th cell subsets in squabs, among which the dysbalance between Th17 and Treg cells appeared to be more closely associated with the damage of the intestinal epithelial barrier function in early weaned squabs.