The small intestine is an important digestive organ and plays a vital role in the life of a pig. In this study, we explored the regulatory role and molecular mechanism of pyrroloquinoline quinone (PQQ) on intestinal health and to discussed the interaction between PQQ and vitamin C (VC). A total of 160 healthy piglets weaned at 21 d were randomly divided into four treatment groups according to 2 × 2 factoring. The results showed that dietary PQQ could significantly decrease the levels of plasma globulin, albumin/globulin (A/G), indirect bilirubin (IBIL), blood urea nitrogen (BUN), creatinine (CREA) (P < 0.05 for each), total bilirubin, (TBIL) (P < 0.01), diamine oxidase (DAO) (P < 0.01) and immunoglobulin G (IgG) (P < 0.0001) and increase the levels of immunoglobulin A (IgA) and immunoglobulin M (IgM) (P < 0.0001) in the plasma of weaned piglets. Similarly, dietary VC could significantly decrease the levels of plasma globulin, A/G, DAO (P < 0.05 for each) and IgG (P < 0.0001) and increase the levels of IgA and IgM (P < 0.0001) in the plasma of weaned piglets. In addition, dietary PQQ increased (P < 0.05) the mRNA levels of antioxidant genes (NQO1, UGT1A1, and EPHX1), thereby enhancing (oxidized) nicotinamide adenine dinucleotide (NAD+) concentration and sirtuin 1 (SIRT1) activity in tissues. However, the addition of 200 mg kg-1 VC to the diet containing PQQ reduced most of the effects of PQQ. We further show that PQQ reduced (P < 0.05) the expression of inflammation-related genes (IL-2, IL-6, TNF-α, and COX-2) via the SIRT1/NF-κB deacetylation signaling. In conclusion, our data reveals that PQQ exerts a certain protective effect on the intestines of piglets, but higher concentrations of VC react with PQQ, which inhibits the regulatory mechanism of PQQ.