Berberis empetrifolia is an endemic species from Chile that produces some bioactive compounds, highlighting the alkaloid berberine, which demonstrated antitumor properties. Taking into account the constant plant supply demand required for medicinal use, the establishment of in vitro propagation protocols allows plant multiplication, without affecting natural populations. Moreover, the in vitro germination is an efficient strategy to obtain axenic plants. The aim of this study was to establish the in vitro germination and to characterize seeds morphology and seedling development of B. empetrifolia. After decontamination of seeds, pre-germinative treatments were applied. Seed viability ranged from 75-90% and the embedding curve revealed permeable integument. The germination was characterized as epigeal phanerocotylar type. Seeds not submitted to pre-germinative treatments reached 10-30% germination rate (GR) and showed asynchronous germination. The most efficient pre-germinative treatment was the storage of seeds at 3˚C for 12 days followed by overnight soaking in GA3 solution associated to seed coat removal. This treatment allowed the seed dormancy to be overcome, reaching GR above 95% and resulting in synchronous germination. The in vitro germination protocol established for B.empetrifolia was efficient to produce phenotypically normal axenic seedlings, allowing their use as source of plant material for biotechnological, phytochemical and pharmacological studies.