This study investigates the effects of novel matrine derivatives as heat shock protein 90 (Hsp90) inhibitors on the proliferation, cloning, and migration ability of lung cancer A549 cells along with their mechanisms. In this work, we used matrine as a lead compound with the research objectives of improving its targeting and anti-tumor activity, as well as developing Hsp90 inhibitors with novel structures. We designed and synthesized 21 matrine derivatives based on the C-14 position of matrine. The antiproliferative activities of all compounds against three types of cancer cells were determined by MTT assay. Most of the compounds showed excellent anticancer activity compared to matrine. Among them, compound H10 showed the most potent antiproliferative activity with IC values of 4.541 ± 0.56 µM (MDA-MB-231), 6.784 ± 0.92 µM (Huh-7), and 3.585 ± 0.45 µM (A549), respectively. Compound H10 not only effectively suppresses the colony formation and migration of A549 cells but also significantly induces cell apoptosis. Subsequent, the impact of compound H10 on Hsp90 protein and proteins associated with the PI3K/Akt/mTOR signaling pathway was evaluated using Western blot analysis. The findings demonstrated that compound H10 effectively suppresses the expression of Hsp90 and induces apoptosis in lung cancer cells by inhibiting the PI3K/Akt/mTOR signaling pathway. This study indicated that compound H10 might serve as a lead compound of Hsp90 inhibitors for the treatment of lung cancers.