Technical Abstract: Saponins are naturally occurring metabolites present in Yerba mate (Ilex paraguariensis), and other plant sources which have been associated with several health benefits. Mate saponins were extracted with methanol from dry leaves, partially purified and quantified. UV-HPLC analysis showed that the methanol extract contained between 10 – 15 mg/g total saponins, predominantly matesaponins 1 and 2. HPLC and LC/ESI-MS-MS were used to identify the saponins in six preparative chromatographic fractions (A, B, C, D, E, and F) obtained from the methanol extract. The major saponins were identified as matesaponin 1 [M-H]- ‘1 and matesaponin 2 [M-H]- _57, with trace amounts of matesaponin 3 [M-H]- _73, matesaponin 4 [M-H]- _19, and matesaponin 5 [M-H]- _83. There were two isomeric forms of matesaponin 2. The extracts were assessed for their in vitro anti-inflammatory effects on macrophages (RAW 264.7). Fractions A, B, and C did not reduce inflammatory markers. Fractions D, E, and F significantly inhibited iNOS (IC35 = 36.3, 29.5, 43.7 µM), PGE2 (IC35 = 23.1, 22.3, 11.7 µM) and COX-2 (IC35 = 45.7, 32.4, 17.0 µM), and were shown to contain matesaponins by mass spectra. Treatments with fraction F (25 µM) resulted in reduction of LPS-induced nuclear translocation of nuclear factor-'B (NF-'B) subunits p50 (49.8%) and p65 (49.0%) indicating that mate fractions inhibit inflammation through NF-'B pathways. Fraction F induced apoptosis through suppression of Bcl-2 and increased Bax protein expressions and activated caspase-3 activity. Saponins from mate leaves may be beneficial to prevent inflammation, thus reducing the risk of colon cancer.