Flammulina velutipes (F. velutipes) polysaccharides have been shown to play a crucial role in preventing inflammatory diseases. However, further exploration is needed to understand the specific relationship between their structure and anti-inflammatory activity. In this study, four structurally distinct F. velutipes polysaccharides (FVP2, FVP2U1, FVP2U2, and FVP2U3) were isolated via a previously established ultrasonic degradation model, and the exploration on the impact of chain conformation on their anti-inflammatory activities was focused. The results indicated that FVP2 exhibited a spherical conformation, while FVP2U1 and FVP2U2 displayed flexible chains, and FVP2U3 showed a semi-rigid chain. The study on the impact of four kinds of F. velutipes polysaccharides on inflammatory response in LPS-stimulated RAW264.7 cells revealed that ultrasonic degradation enhanced the anti-inflammatory activity of FVP2. The primary anti-inflammatory mechanism involved the inhibition of NO, TNF-α, IL-1β, and IL-6 release, and reduced transcriptional levels of TLR4, MyD88, NF-κB, and NLRP3. Among the ultrasonically degraded polysaccharides with compliant chain conformations and lower molecular weight demonstrated superior anti-inflammatory activity. These results provide insights into the relationship between molecular weight, chain conformation, and anti-inflammatory activity of F. velutipes polysaccharides, contributing to the potential application of ultrasound in developing highly active anti-inflammatory polysaccharides and related nutritional products.