Smilax glabra Roxb. has been used for a long time as both food and folk medicine. In the present study, phenolic-enriched extract of S. glabra (PEESG) was extracted with 70% ethanol and purified by HP-20 column chromatography. Its antioxidant and anti-inflammatory activities were evaluated by radical scavenging assay, reducing power determination, and lipopolysaccharide (LPS)-induced RAW264.7 cells assays, respectively. PEESG exhibited obviously scavenging capacity for DPPH and ABTS radicals, as well as significant reducing power for ferric ion. Particularly, PEESG (12.5-50 μg/mL) showed a significantly higher efficiency for scavenging ABTS than that of ascorbic acid and no significant difference with ascorbic acid for DPPH scavenging. PEESG also possessed a significant suppression effect on proinflammatory mediators production, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), in LPS-induced RAW264.7 cells. In addition, the main ingredients of PEESG were identified using ultrahigh pressure liquid chromatography coupled to electrospray mass spectrometry (U-HPLC-ESI-MS). Seventeen components, including 5-O-caffeoylshikimic acid, neoastilbin, astilbin, neoisoastilbin, isoastilbin, engetin and isoengeletin were identified. These findings strongly suggest the potential of PEESG as a natural antioxidant and anti-inflammatory agent.