Abstract: OBJECTIVE To investigate the antitumor activity and mechanism of ciprofloxacin combined with RC48 which is an anti-HER2 antibody-drug conjugate.METHODS CCK-8 assay was used to detect the inhibitory effect of ciprofloxacin, RC48 and their combination(simultaneously or sequentially with different proportions) on the proliferation of HER2 positive tumor cells. The combined index method was used to evaluate the combined effect of the two drugs.RESULTS RC48 displayed a significant inhibitory effect on the proliferation of SK-BR-3 and NCI-N87 cells with the IC₅₀ values of(4.91±1.15)ng·mL^–1and (14.54+0.85)ng·mL^–1respectively, and the maximum inhibition rates were 87.2% and 82.3% respectively. Ciprofloxacin alone had no significant antitumor effect in the dose range of 234.38-7 500 ng·mL^–1. When it was given with RC48 at the same time, the anti-tumor effect of RC48 was significantly reduced(P<0.05) and the two drugs showed an antagonistic effect. However, when it was used successively with RC48 was administered first and then ciprofloxacin, with an interval of 6-48 h, the antitumor activity increased significantly(P<0.05). The two drugs showed synergistic effect, and the synergistic effect was still maintained when the combined efficacy was as high as 95.3%.CONCLUSION Sequential administration of RC48 and ciprofloxacin, at 6-48 h intervals, especially at 6 h intervals, can enhance the anti-tumor efficacy, and the two drugs show a synergistic effect.