The present study was conducted to determine whether dietary threonine supplementation can improve immunity of weaned pigs challenged by swine Pseudorabies live vaccine (SPLV). Thirty crossbred piglets weaned at 21 days of age were randomly assigned to three groups receiving diets containing true ileal digestible threonine (TIDT) at 0.74, 0.89 and 1.11% for 14 days. On day 8, all pigs were injected intramuscularly with SPLV or sterile 0.9% NaCl solution. SPLV injection enhanced serum IgA, IgM, IgG, IFN-γ, IL-1β, TNF-α and IL-10 concentrations (p < 0.05) and stimulated the relative mRNA abundance of Toll-like receptors (TLR3, TLR7 or TLR9) in different tissues (p < 0.05). Under no challenge, increasing dietary TIDT levels enhanced serum IgG (p < 0.05), IgM (p = 0.07) and IFN-γ (p < 0.05) concentration, tended to decrease serum IL-1β, TNF-α and IL-10 concentration, and regulated relative mRNA abundance of TLR3, TLR7 or TLR9 in different tissues (p < 0.05). However, there was a synergistic role for increasing the serum IL-10 concentration between dietary TIDT levels and SPLV injection (p < 0.05). Under SPLV challenge, increasing dietary TIDT levels attenuated the increase of the serum IFN-γ concentration, and the increase of the relative mRNA abundance of TLR3, TLR7 and TLR9 in the different tissues (p < 0.05). These results suggest that an appropriate dietary threonine supplementation could improve the immune status of weaned pigs injected with SPLV by down-regulating the expression of TLR3, TLR7 and TLR9 in tissues, and thus regulating T-helper cytokine secretion.