Arginine supplementation improves intestinal damage and intestinal immunity, but the underlying mechanism of the effects of arginine supplementation on intestinal SIgA secretion is largely unknown. Therefore, this study was conducted to investigate the underlying pathway on the effects of arginine supplementation in secretory IgA (SIgA) production in mice. The results showed that 0.4% arginine supplementation promoted (P < 0.05) SIgA production in intestinal lumina and IgA plasma cell numbers in the ileum of mouse. Arginine supplementation significantly increased (P < 0.05) cytokines expression in mouse ileal associated with T cell-dependent and T cell-independent pathways of SIgA induction, including IL-5, IL-6, IL-13, transforming growth factor (TGF-)β2, TGF-β3, TGF-βR2, a proliferation-inducing ligand (APRIL), B cell-activating factor (BAFF), vasoactive intestinal peptide (VIP) receptor, and retinal dehydrogenases. Further study showed that 0.4% arginine supplementation markly decreased (P < 0.05) bacterial loads in mouse mesenteric lymph nodes and increased bacterial invasion into the mouse ileal wall, while supplementation of antibiotic abrogated the influence of arginine supplementation on SIgA secretion. Therefore, these data suggest that arginine supplementation might promote SIgA secretion through cytokines and intestinal microbiota might play an important role in SIgA secretion by arginine supplementation in the mouse intestine.