Abstract: To explore new agents of quinolone derivatives with high activity against Gram-positive and Gram-negative microorganisms, 7-(3-amino-4-alkoxyimino-1-piperidyl) quinolones were designed and synthesized, and their activity against Gram-positive and Gram- negative microorganisms were tested in vivo and in vitro. Twenty one target compounds were obtained. Their structures were established by ¹H NMR, HRMS and X-ray crystallographic analysis. The target compounds possess different antimicrobial activities against both Gram-negative and Gram-positive microorganisms. Compounds 14a and 14m have broad spectral antibacterial activities. They show better antibacterial activities against 12 strains Gram-positive bacteria than three references. In particular, their activities against S.aureus and S.epidermidis (including MRSA and MRSE) were 4-16 times than that of gemifloxacin and balofloxacin, and 8-64 times than that of levofloxacin. The MIC values to S.aureus strains of compounds 14a and 14m were 0.25-1 mg·L^-1 and 0.125-1 mg·L^-1, to S.epidermidis strains were 0.5-4 mg·L^-1 and 1-8 mg·L^-1, respectively. The in vivo results showed that they have as good internal protection as gemifloxacin and moxifloxacin against systemic infection model in mice (P>0.05).