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Abstract Background and purpose To compare organ-at-risk doses and setup reproducibility using the prone and supine orientations in volumetric modulated arc therapy (VMAT) for rectal cancer. Materials and methods Seventeen consecutive rectal cancer patients undergoing preoperative radiation were selected and setup in either the prone (N = 8) or supine (N = 9) position. All patients were treated using posteriorly-applied VMAT. Bladder and small bowel dose and cone beam CT (CBCT) reproducibility metrics were retrospectively collected. Results Dose metrics for bladder and small bowel did not show significant differences between the prone and supine orientations. The prone data had a trend for smaller irradiated volumes than supine for the small bowel at lower doses—V20 (prone: 135 ± 99 cm3; supine: 201 ± 162 cm3) and V30 (prone: 78 ± 71 cm3; supine: 105 ± 106 cm3). At higher doses, the trend reversed as exemplified by the small bowel V50.4 (prone: 20 ± 28 cm3; supine: 10 ± 14 cm3). CBCT data showed that rotational errors in pitch and roll were significantly larger for the prone vs. supine orientation (pitch: 2.0° ± 1.3° vs. 0.8° ± 1.1° p