Vacation has been shown to be an important restorative activity in the general population; less is known about physicians' vacation behaviors and their association with burnout and professional fulfillment. To examine the number of vacation days taken per year and the magnitude of physician work while on vacation and their association with physician burnout and professional fulfillment, by individual and organizational characteristics. This cross-sectional survey of US physicians was conducted between November 20, 2020, and March 23, 2021. Data analysis was performed from March to July 2023. Burnout was measured using the Maslach Burnout Index, and professional fulfillment was measured using the Stanford Professional Fulfillment Index. Number of vacation days taken in the last year, time spent working on patient care and other professional tasks per typical vacation day (ie, work on vacation), electronic health record (EHR) inbox coverage while on vacation, barriers to taking vacation, and standard demographics were collected. Among 3024 respondents, 1790 of 3004 (59.6%), took 15 or fewer days of vacation in the last year, with 597 of 3004 (19.9%) taking 5 or fewer days. The majority, 2104 respondents (70.4%), performed patient care-related tasks on vacation, with 988 of 2988 (33.1%) working 30 minutes or more on a typical vacation day. Less than one-half of physicians (1468 of 2991 physicians [49.1%]) reported having full EHR inbox coverage while on vacation. On multivariable analysis adjusting for personal and professional factors, concern about finding someone to cover clinical responsibilities (odds ratio [OR], 0.48 [95% CI, 0.35-0.65] for quite a bit; OR, 0.30 [95% CI, 0.21-0.43] for very much) and financial concerns (OR, 0.49 [95% CI, 0.36-0.66] for quite a bit; OR, 0.38 [95% CI, 0.27-0.54] for very much) were associated with decreased likelihood of taking more than 3 weeks of vacation per year. Taking more than 3 weeks of vacation per year (OR, 0.66 [95% CI, 0.45-0.98] for 16-20 days; OR, 0.59 [95% CI, 0.40-0.86] for >20 days vs none) and having full EHR inbox coverage while on vacation (OR, 0.74; 95% CI, 0.63-0.88) were associated with lower rates of burnout on multivariable analysis, whereas spending 30 minutes or longer per vacation day on patient-related work (OR, 1.58; 95% CI, 1.22-2.04 for 30-60 minutes; OR, 1.97; 95% CI, 1.41-2.77 for 60-90 minutes; OR, 1.92; 95% CI, 1.36-2.73 for >90 minutes) was associated with higher rates of burnout. In this cross-sectional study of 3024 physicians, the number of vacation days taken and performing patient-related work while on vacation were associated with physician burnout. System-level efforts to ensure physicians take adequate vacation and have coverage for clinical responsibilities, including EHR inbox, may reduce physician burnout.