This work aims to compare the chemical composition and anti-inflammatory effects on RAW264.7 macrophages of Keemun black tea stems and leaves. A total of 50 volatile compounds were identified in tea stems and leaves, and aldehydes, alcohols, and esters were the main volatile compound categories. There were 11 key volatile compounds, including geraniol, benzeneacetaldehyde, methyl salicylate, linalool, etc. contributed to distinguishing the tea stems from the tea leaves. In the quantitative and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics analysis, higher contents of amino acids, monosaccharides, and quinic acids were found in stems than those in leaves. Inversely, higher contents of tea pigments, flavan-3-ols, gallic acid, purine alkaloids, and flavonol glycosides were present in tea leaves than in stems. LC-MS-based metabolomics also revealed that organic acids were the most critical non-volatile compounds responsible for the differences between tea stems and leaves. Furthermore, tea stems had better inhibiting effects of pro-inflammatory cytokines (interleukin (IL)-1β and IL-6) in lipopolysaccharide-challenged RAW264.7 macrophages than tea leaves, while no significant differences exist between leaves and stems for inhibiting the secretion of tumor necrosis factor α (TNF-α) and NO. In conclusion, our results support using Keemun black tea stems as a novel source of anti-inflammatory compounds.