Physician wellness is vital to career satisfaction, provision of high quality patient care, and the successful education of the next generation of physicians. Despite this, the number of physicians experience symptoms of burnout is rising. To assess the impact of organizational culture on physicians' professional fulfillment and burnout, we surveyed full-time Department of Medicine members at the University of Toronto. A cross-sectional survey assessed: physician factors (age, gender, minority status, disability, desire to reduce clinical workload); workplace culture (efforts to create a collegial environment, respectful/civil interactions, confidence to address unprofessionalism without reprisal, witnessed and/or personally experienced unprofessionalism); professional fulfillment and burnout using the Stanford Professional Fulfillment Index. We used multivariable linear regression to examine the relationship of measures of workplace culture on professional fulfillment and burnout (scores 0-10), controlling for physician factors. Of 419 respondents (52.0% response rate), we included 400 with complete professional fulfillment and burnout data in analyses (60% ≤ age 50, 45% female). Mean scores for professional fulfillment and burnout were 6.7±1.9 and 2.8±1.9, respectively. Controlling for physician factors, professional fulfillment was associated with satisfaction with efforts to create a collegial environment (adjusted beta 0.45, 95% CI 0.21 to 0.70) and agreement that colleagues were respectful/civil (adjusted beta 0.85, 95% CI 0.53 to 1.17). Lower professional fulfillment was associated with higher burnout scores. Controlling for professional fulfillment and physician factors, lower confidence in taking action to address unprofessionalism (adjusted beta -0.22, 95% CI -0.40 to -0.03) was associated with burnout. Organizational culture and physician factors had an impact on professional fulfillment and burnout. Professional fulfillment partially mediated the relationship between organizational culture and burnout. Strategies that promote inclusion, respect and civility, and safe ways to report workplace unprofessionalism are needed in academic medicine.