This study aimed to investigate the anti-inflammatory effects of Prunus cerasifera Ehrhart extract (PCEE) on lipopolysaccharide (LPS)-stimulated RAW264.7 cells and to elucidate its underlying molecular mechanism. First, PCEE was fractionated by gradient elution with ethanol, and the separated fractions were evaluated for total polyphenol and flavonoid contents, as well as antioxidant activity. Next, the 70% ethanol-eluted fraction (PCEE-70) was selected to measure its effects on biochemical indexes related to antioxidant defense and oxidative stress in LPS-stimulated RAW264.7 cells. Finally, Western blot (WB) was employed to detect the expression levels of phosphorylated and non-phosphorylated proteins associated with the nuclear factor kappa B (NF-κB) p65 and phosphoinositide 3-kinase/protein kinase B/c-Jun N-terminal kinase (PI3K/AKT/JNK) signaling pathways. The experimental results demonstrated that PCEE-70 had higher contents of polyphenols and flavonoids along with stronger antioxidant activities compared to the other fractions. PCEE-70 significantly inhibited the levels of nitric oxide (NO), reactive oxygen species (ROS) and malondialdehyde (MDA), while enhancing the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). It also reduced the levels of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor-α(TNF-α). Furthermore, WB results indicated that PCEE-70 significantly downregulated the expression of TNF-α, p-NF-κB p65, p-IκB, p-PI3K, p-AKT, and p-JNK proteins. In conclusion, PCEE-70 can inhibit the secretion of inflammatory factors in LPS-induced RAW264.7 cells, thereby mitigating oxidative stress-induced cellular damage. Its action mechanism is associated with the suppression of NF-κB and PI3K/AKT signaling pathway activation.