<jats:title>Abstract</jats:title><jats:p>Chemical studies of the Chinese herb <jats:italic>Corydalis saxicola</jats:italic> <jats:sc>Bunting</jats:sc> led to the isolation and identification of 14 alkaloids, <jats:bold>1</jats:bold>–<jats:bold>14</jats:bold>. Seven of these compounds, <jats:bold>4</jats:bold>–<jats:bold>9</jats:bold> and <jats:bold>11</jats:bold>, were obtained from this plant for the first time. Feruloylagmatine (<jats:bold>7</jats:bold>) is the first guanidine‐type alkaloid to be identified in the family Papaveraceae and in dicotyledonous plants. All of the isolated compounds were assayed for inhibitory activity against human DNA topoisomerase I. A DNA cleavage assay demonstrated that these alkaloids specifically inhibit topoisomerase through stabilization of the enzyme–DNA complex. Among the isolated alkaloids, (−)‐pallidine (<jats:bold>8</jats:bold>) and (−)‐scoulerine (<jats:bold>11</jats:bold>) showed strong inhibitory activities toward topoisomerase I that were comparable to camptothecin, a typical topoisomerase I inhibitor. A preliminary structure–activity relationship study suggested that the quaternary ammonium ion might play an important role in topoisomerase I inhibition by the isoquinoline alkaloids. These data indicated that DNA topoisomerase I inhibition represents probably one of the anticarcinogenic mechanisms of <jats:italic>C. saxicola.</jats:italic>