Early postnatal life represents a critical window of GI immune development when lifelong immune function is shaped. Yet, little is known on how early life events impact long-term immune development in animals and people. Based on our previous research, we investigated how biological sex and early weaning (EW) stress interact to shape long-term GI immune development in the pig. Female (F), male intact (MI) and male castrated (MC) piglets (Yorkshire-cross) were weaned at 17 d (EW) or at 26 d of age (Later weaning; LW). At 77 d of age, tissue samples were harvested for RNA-seq, qPCR, and immunoassays. Serum cytokine and ileal mucosal IgG responses were measured following oral and intramuscular vaccination for Lawsonia intracellularis (LI) and PCV2, respectively. Compared with LW pigs, EW MC and MI pigs had reduced body weight gain (P< 0.05). The impact of biological sex and EW were observed in vaccine responses. Serum from F showed higher PCV2-specific IgG titers and LI-antibody levels compared with MC and MI pigs. EW F had higher IgG titers in response to vaccination compared with LW F. In contrast, EW MC had suppressed antibody titers compared with LW MC. Pathway analysis of RNA-seq data from ileal mucosa revealed enrichment of genes associated with increased in immune-related pathways (eg inflammation) in EW F. In contrast, EW MC exhibited enrichment for genes associated with a down-regulation of immune response (eg immune cell migration). Together, these studies showed that biological sex and EW have a major impact on GI immune development in the pig. An understanding of the role and underlying sex-specific mechanisms driving altered immune development in EW pigs could reveal new targets for enhancing early and long-term immune development.