The search for new plant-based anti-inflammatory drugs continues in order to overcome the detrimental side effects of conventional anti-inflammatory agents, both steroidal and nonsteroidal. This study involves the quinoline SPE2, 7-hydroxy-6-methoxyquinolin-2(1 H)-one, isolated from the EtOAc fraction of Spondias pinnata bark. Structure elucidation was done using analytical spectroscopic methods including Fourier transform infrared spectroscopy, high-resolution electrospray ionization mass spectrometry, nuclear magnetic resonance spectroscopy, and single-crystal X-ray crystallography. The anti-inflammatory activity of SPE2 was evaluated in a lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 model. SPE2 effectively suppressed LPS-induced overproduction of pro-inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, and reactive oxygen species. Expression levels of NO synthesizing enzyme, cyclooxygenase-2, TNF-α, IL-6 and IL-1β were also determined to return to normal after SPE2 treatment. Localization of NF-κB was evaluated by confocal microscopy and Western blotting, which showed a dose-dependent reduction of NF-κB inside the nucleus and an increase in cytoplasmic NF-κB with SPE2 treatment. Collectively, the results suggest that SPE2 has anti-inflammatory activity via inhibition of NF-κB activation.