<jats:title>Abstract</jats:title><jats:p>Three new cytochalasins Z<jats:sub>21</jats:sub>–Z<jats:sub>23</jats:sub> (<jats:bold>1</jats:bold>–<jats:bold>3</jats:bold>, resp.), together with five analogs, <jats:bold>4</jats:bold>–<jats:bold>8</jats:bold>, were isolated from <jats:italic>Spicaria elegans</jats:italic> KLA03 by the OSMAC (one strain‐many compounds) approach with adding <jats:sc>L</jats:sc>‐ and <jats:sc>D</jats:sc>‐tryptophan during its cultivation. The structures of new cytochalasins were elucidated on the basis of comprehensive 1D‐ and 2D‐NMR and HR‐ESI‐MS analyses. Cytochalasins Z<jats:sub>21</jats:sub> and Z<jats:sub>22</jats:sub> (<jats:bold>1</jats:bold> and <jats:bold>2</jats:bold>, resp.), and compound <jats:bold>5</jats:bold> showed cytotoxic activities against A‐549 cell lines with <jats:italic>IC</jats:italic><jats:sub>50</jats:sub> values of 8.2, 20.0, and 3.1 μ<jats:sc>M</jats:sc>, respectively.