BACKGROUND Nurses and physicians are key members of healthcare teams. While physicians are responsible for the diagnosis and treatment of patients, nurses are part of the treatment and the primary practitioners of patient care. Nurses' professional autonomy, collaboration with physicians, and practice behaviors in treatment and patient care practices are interrelated. OBJECTIVE In the present study, we examined the mediating effect of physician-nurse collaboration on the relationship between nurses' practice behaviors and their professional autonomy. METHODS The present study utilized a cross-sectional survey design following quantitative methods. METHODS This study was conducted in the Istanbul Province of Turkiye from September to October 2022. The sampling method used was a convenience sampling strategy to provide easier access to participants when selecting nurses from different health institutions. The mean age of the 295 nurses was 31.23 years, with ages ranging from 21 to 59 years. The data analysis was conducted using IBM's SPSS 24.0 software package and the Process Macro 4.0 plug-in. Research ethics approval was obtained from the researcher's university. RESULTS Physician-nurse collaboration is positively associated with practice behaviors and professional autonomy. Nurses' professional autonomy in practice behaviors through physician-nurse collaboration is significant (95% CI [0.043, 0.135]). CONCLUSIONS Our results revealed the relationships among physician‒nurse collaboration, professional autonomy, and practice behaviors among nurses. CONCLUSIONS Our results provide evidence on the underlying factors of nurses' practice behaviors in patient care and guide the development of an intervention program to enhance this collaboration. Hospital managers can contribute to a collaborative physician‒nurse working environment.