The flavonoid constituents of Aesculus wilsonii, a source of the Chinese medicinal drug Suo Luo Zi, and their in vitro anti-inflammatory effects were investigated. Fifteen flavonoids, including aeswilflavonosides IA–IC (1–3) and aeswilflavonosides IIA–IIE (4–8), along with seven known derivatives were isolated from a seed extract. Their structures were elucidated by extensive spectroscopic methods, acid and alkaline hydrolysis, and calculated electronic circular dichroism (ECD) spectra. Among them, compounds 3 and 7 possess a 5-[2-(carboxymethyl)-5-oxocyclopent-yl]pent-3-enylate or oleuropeoylate substituent, respectively, which are rarely reported in flavonoids. Compounds 2, 3, 7, and 1215 were found to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cell lines. In a mechanistic assay, the flavonoid glycosides 2, 3, and 7 reduced the expressions of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) induced by LPS. Further investigations suggest that 2 and 3 down-regulate the protein expression of TNF-α and IL-6 by inhibiting the phosphorylation of p38. Compound 7 was found to reduce the production of inducible nitric oxide synthase (iNOS), and the secrection of TNF-α and IL-6 through inhibiting nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) signaling pathway. Compounds 2, 3 and 7 possessed moderate inhibitory activity on the expression of signal transducer and activator of transcription 3 (STAT3). Taken together, the data indicate that the flavonoid glycosides of A. wilsonii seeds exhibit NO release inhibitory activity through mitogen-activated protein kinase (p38) [MAPK (p38)], NF-κB and STAT3 cross-talk signaling pathways.