The Mediterranean buckthorn, Rhamnus alaternus L., is a plant used in traditional medicine in Mediterranean countries. We aimed at characterizing its phenolic compounds and explore potential antihyperlipidemic activity of this plant. The profile of phenolic compounds in R. alaternus leaf crude methanolic extract (CME) and its liquid-liquid extraction-derived fractions were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS²). Effects of CME on: circulating lipids in rats with Triton WR-1339-induced hyperlipidemia, intracellular lipid accumulation and expression of genes of fatty acid metabolism in human hepatoma HepG2 cells, and adipogenesis in the 3T3-L1 murine adipocyte cell model were assessed. The HPLC/ESI-MS² analytical profile revealed a total of fifteen compounds, of which eleven were identified. Oral CME administration decreased blood levels of cholesterol and triacylglycerols in hyperlipidemic rats (by 60% and 70%, respectively, at 200 mg CME/kg). In HepG2 cells, CME exposure dose-dependently decreased intracellular lipids and up-regulated gene expression of carnitine palmitoyltransferase 1 involved in fatty acid oxidation. In the 3T3-L1 model, CME favored preadipocyte proliferation and adipogenesis, pointing to positive effects on adipose tissue expandability. These results suggest novel uses of R. alaternus by showing that its leaves are rich in flavonoids and flavonoid derivatives with an antihyperlipidemic effect in vivo and in hepatic cells.