Abstract <p><b>Objective:</b> This article focuses on the design, synthesis, and characterization of C-19 acylamino-functionalized isosteviol derivatives, and their evaluation for LSD1 inhibitory activity as well as molecular docking studies. <b>Methods:</b> The novel C-19 acylamino-functionalized isosteviol derivatives were designed based on the principle of drug combination, and were subsequently synthesized <i>via</i> acetylation with acyl chlorides and condensation with amines. The LSD1 inhibitory activity of the synthesized compounds was evaluated using the LSD1 small molecule inhibitor screening platform. Molecular docking studies were performed using MOE (Version 2019). <b>Results and Discussion:</b> The synthesized C-19 acylamino-functionalized isosteviol derivatives were characterized by IR, NMR, and HR-MS spectra. The results of the anti-LSD1 activity assays showed that compounds with cyclic substituents generally exhibited excellent inhibitory activity. In particular, compound <b>IIIl</b> exhibited the best LSD1 inhibitory effect, with an IC₅₀ value of 8.523 ± 0.882 μM. Further molecular docking studies revealed that the 16-carbonyl group of compound <b>IIIl</b> formed a hydrogen bond with the Ser289 residue, and its aromatic ring formed a π-H interaction with the Met332 residue. Additionally, the top-ranked docking pose of compound <b>IIIl</b> showed a strong binding affinity to the LSD1 protein, with a docking score of –5.273.<b>Conclusions:</b> This study lays the groundwork for the development and structural modification of new isosteviol-based drugs.</p>