ObjectiveTo study the effects of matrine derivative C4 on migration， invasion and apoptosis of non-small cell lung cancer cells by targeting heat shock protein 90. MethodMolecular docking and Western blot were used to detect the regulation of matrine derivative C4 （0， 1， 2， 4， 8 μmol·L^-1） on heat shock protein 90 （Hsp90）. methyl thiazolyl-tetrazolium （MTT） assay was conducted to observe the effects of C4 （0， 0.25， 0.5， 1， 2， 4， 8， 16 μmol·L^-1） on viability of A549 cells. The impacts of different concentrations of C4 （0， 2， 4， 8 μmol·L^-1） on the proliferation， migration， invasion and apoptosis of A549 cells were explored by clone formation assay， wound healing assay， Transwell assay， and flow cytometry， respectively. Western blot was employed to detect the effects of C4 （0， 1， 2， 4， 8 μmol·L^-1） on the protein expression of A549 protein kinase B （Akt）， phosphorylated protein kinase B （p-Akt）， epidermal growth factor receptor （EGFR）， phosphatidylinositol 3 kinase （PI3K）， p-PI3K， cysteine aspartate protease 9 （Caspase-9）， B-cell lymphoma 2 （Bcl-2）-associated X （Bax）， Bcl-2， and Bcl-2-associated agonist of cell death （Bad）. ResultMatrine derivative C4 could effectively interact with Hsp90 protein through a water-mediated hydrogen bond network with residues of asparagine-51 （ASN-51）， glycine-135 （GLY-135）， and phenylalanine-138 （PHE-138）， and the π-π stacking interaction with the phenyl ring of PHE-138 contributed to its affinity. In addition， compared with blank group， C4 down-regulated the protein expression of Hsp90 in A549 cells （P<0.05， P<0.01）， and reduced the cell viability （P<0.05， P<0.01）. The half-maximal inhibitory concentrations （IC₅₀） of C4 on A549 cells were （12.32±0.14）， （7.79±0.16） and （3.26±0.09） μmol·L^-1 at 24， 48 and 72 h， respectively. Compared with the conditions in the blank group， the clone formation ability of A549 cells in C4 （2， 4， 8 μmol·L^-1） groups was decreased （P<0.05， P<0.01） in a concentration-dependent manner， and C4 inhibited the migration and invasion of A549 cells （P<0.05， P<0.01）， with the most significant inhibition observed at 8 μmol·L^-1 （P<0.01）. Moreover， C4 induced apoptosis of A549 cells （P<0.01）， and the apoptosis rates at 0， 2， 4 and 8 μmol·L^-1 were （2.28±0.35）%， （4.97±0.36）%， （8.86±0.50）% and （20.67±0.99）%， respectively. It also up-regulated the protein expression of Caspase-9， Bax and Bad （P<0.05， P<0.01）， while down-regulated those of PI3K， p-PI3K， p-Akt， EGFR and Bcl-2 to varying degrees （P<0.05， P<0.01）， and the protein expression level of Akt did not change significantly. ConclusionMatrine derivative C4 played an anti-tumor role by inhibiting cell viability， proliferation， migration and invasion and promoting apoptosis response， which might be realized by inhibiting Hsp90/PI3K/Akt signaling pathway.