Abstract: This study aimed to isolate and prepare highly purified impurity C from doxycycline hyclate by a preparative HPLC method and to inspect the toxicity and in vitro antimicrobial activity of the impurity C of doxycycline hyclate. The solution of doxycycline hyclate treated with heat produced a solution containing 10% of impurity C which was firstly separated by the Sapphire C₁₈(21. 2 mm×250 mm, 5 μm)column with 0. 1% acetic acid-acetonitrile(83 ∶17)as the mobile phase at 20 mL/min. Secondly, rotary evaporation of the eluted solution at the time of 8. 4 min was performed at 50 °C to remove organic solvent. Then the target product was prepared after freeze drying of evaporated solution adjusting pH to 1. 8 with formic acid. The target product was identified with ultraviolet absorbance(UV), infrared(IR), mass spectrometry(MS)and nuclear magnetic resonance(NMR), and its purity was be determined by HPLC. Meanwhile, cytotoxicity and genotoxicity in the Chinese hamster lung cells, toxicity on the development of zebrafish embryos and in vitro antimicrobial activity were compared among impurity C of doxycycline hyclate, doxycycline, metacycline and β-doxycycline. Results showed that prepared product was confirmed to be the impurity C of doxycycline hyclate. Its purity was 90. 1%, which had been the highest so far. In the cellular toxic tests and genetic toxic tests of Chinese hamster lung cells, impurity C of doxycycline hyclate, doxycycline, metacycline and β-doxycycline were somewhat toxic to Chinese hamster lung cells. Toxicity gradually decreased from doxycycline, impurity C of doxycycline hyclate, β-doxycycline to metacycline from -S₉mix test results; toxicity gradually decreased from doxycycline, β-doxycycline, impurity C of doxycycline hyclate to metacycline from +S₉mix test results; the aberration rate of all the tested related substances was less than 5%, and no obvious genotoxicity was found. According to test results of the development of zebrafish embryos, impurity C of doxycycline hyclate showed the strongest teratogenicity and lethality. Invitro antimicrobial tests revealed that impurity C of doxycycline hyclate had a weaker antimicrobial activity, and invitro antimicrobial activity potential of the tested compounds followed the order: metacycline, doxycycline, impurity C of doxycycline hyclate, β-doxycycline. Studies on safety and effectiveness indicated that impurity C of doxycycline hyclate belonged to toxic and ineffective impurity and need to be controlled individually in quality standard. A useful suggestion was given to revise the quality standard of doxycycline hyclate and its preparation in the current Pharmacopoeia of the People′s Republic of China.