Background: Nowadays, irrational use of antimicrobials has threatened public health. It’s necessary to expanding the use of clinical practice guideline (CPG) on antimicrobial for facilitating the proper use of antimicrobial. However, the utilization status of CPG on antimicrobial and the influencing factors are largely unknown. Methods: A cross-sectional survey was conducted, using a structured questionnaire, on a sample among physicians from 16 public hospitals in eastern, central and western part of China. A multilevel regression model was employed to examine factors associated with physicians’ utilization of CPG on antimicrobial. Results: A total of 815 physicians included in this study. About eighty percent of the surveyed physicians reported their strict adherence to the CPG on antimicrobial. Dimensions of “subjective norm”, “perceived risk” and “behavioral intention” from the domain of physician belief, dimension of “ease of use” from the domain of CPG traits, and dimensions of “top management support” and “organization & implementation” from the domain of hospital practice were significantly associated with physicians’ utilization of CPG on antimicrobial. And most demographics of physician were not found to be significantly related to the CPG use. In addition, results showed region is a significant factor affecting physicians’ CPG use. Conclusions: This study depicted the current status of CPG on antimicrobial and comprehensively identified its potential determinants not only from the three domains, such as physician belief, at the individual level, but also from the location region at the organizational level. The results will provide direct reference on implementation of CPG on antimicrobial and will be generalizable to the setting of health care system.