Background: The early postnatal period is critical for immunity, and feeding docosahexaenoic acid (DHA) has been demonstrated to affect immune development. Objective: The objective of this study was to determine the importance of feeding DHA during suckling and/or weaning on immune function and oral tolerance (OT). Methods: Sprague-Dawley rats were randomly assigned to 1 of 2 nutritionally adequate diets throughout lactation (21 d): a control (n = 12, 0% DHA) diet or a DHA (n = 8, 0.9% DHA) diet. At 11 d, suckled pups from each dam were randomly assigned to a mucosal OT challenge: placebo or ovalbumin. At week 5, all pups systemically received ovalbumin + adjuvant to induce systemic immunization. At 21 d, pups from each dam were randomly assigned to 1 of the 2 diets for 21 d in a factorial design after which immune function and OT were assessed. Results: Feeding dams DHA during lactation resulted in a 40-60% higher splenocyte production of interleukin (IL)-10 when stimulated with concanavalin A, lipopolysaccharide (LPS), or ovalbumin and a 100% higher production of interferon (IFN)-gamma with LPS (P< 0.05) than feeding the control diet to the pups. In comparison with pups fed the control diet, feeding DHA at weaning resulted in a 25% lower type 1 T helper (IL-1 beta) and type 2 T helper (IL-6) response by splenocytes after LPS stimulation and a 33% lower plasma concentration of ovalbumin-specific immunoglobulin (Ig) G (P < 0.05). Pups that did not receive additional DHA during the study had a 70% higher plasma concentration of ovalbumin-specific IgE than did the pups that received DHA at suckling and/or weaning (P< 0.05). Conclusions: Feeding additional DHA during suckling had a beneficial programming effect on the ability of immune cells to produce IFN-gamma and IL-10, and feeding DHA during weaning resulted in a lower inflammatory response. Providing no dietary DHA in either of the critical periods of immune development prevented the establishment of OT in female rat offspring.