Lysimachia vulgaris L. (yellow loosestrife) is a medicinal plant that has been used in the treatment of fever, ulcer, diarrhea and wounds in traditional medicine. A reliable in vitro culture protocol for yellow loosestrife was established. Explants (leaf lamina, stem internode and root segments) were cultured on Murashige and Skoog minimal organics (MSMO) medium supplemented with various plant growth regulator combinations. Of the tested combinations, those involving benzyladenine (BA) with either indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) were the most effective for all used explants in shoot production. Best shoot proliferation was obtained from leaf lamina explant cultured on media containing 0.5 mg/l BA and 0.1, 0.5 or 1 mg/l IBA, from stem internode explant cultured on media containing 1 mg/l BA and 0.5 mg/l IBA or 0.01 mg/l thidiazuron (TDZ) and 0.5 mg/l IAA, and from root explant cultured on media containing 0.5 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were rooted on MSMO medium containing different concentrations of IAA, IBA, 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA). IBA was determined as the most effective auxin for rooting. Most shoots developed roots on medium with 0.5 mg/l IBA.