<h3>BACKGROUND CONTEXT</h3> Lateral lumbar interbody fusion (LLIF) was initially developed to be performed in the lateral decubitus position, facilitating indirect neural decompression and interbody cage insertion while obviating the need for paraspinal musculature dissection. Nevertheless, this technique may limit segmental lordotic correction and–when posterior instrumentation is indicated–requires either procedure staging or lateral decubitus placement of pedicle screws. To address these drawbacks, attention has recently shifted toward the prone transpsoas technique. However, anatomical relationships between the vertebral column and adjacent anatomy may be affected by prone positioning. <h3>PURPOSE</h3> To quantify radiographic differences in psoas morphology, lumbar plexus, great vessel anatomy and lumbar lordosis between supine and prone positioning to optimize surgical planning and minimize the risk of neurovascular injury. <h3>STUDY DESIGN/SETTING</h3> Retrospective cohort study. <h3>PATIENT SAMPLE</h3> Consecutive single-surgeon cohort with lumbar degenerative pathologies undergoing posterior spinal fusion between June 2020 and December 2021. Any patient with intraoperative computed tomography (CT) imaging obtained using O-arm spinal navigation while positioned extended-prone on an open Jackson table was included. <h3>OUTCOME MEASURES</h3> Psoas morphology, iliac vasculature anatomy, lumbar lordosis. <h3>METHODS</h3> Measurements were made on supine preoperative magnetic resonance imaging (MRI) and prone intraoperative CT from L2 to L5 levels. These included the anteroposterior and mediolateral proximity of the psoas, aorta, inferior vena cava (IVC), and anterior iliac vessels to the vertebral body. Psoas transverse and longitudinal diameters, psoas cross-sectional area (CSA), total lumbar lordosis, and segmental lordosis were assessed. Supine and prone anatomies were compared via paired t-test, while ANOVA and the Spearman correlation coefficient evaluated the relationship between lumbar level and the magnitude of translation. <h3>RESULTS</h3> Twenty patients were included for analysis. Prone positioning led to significant psoas anterior translation (5.14 mm prone vs. 1.5 mm supine, P < 0.001), although the magnitude of anterior translation significantly decreased at more caudal segments (ρ: -0.39, P = 0.002) and was lowest at L5 where slight posterior retraction was observed (-0.31 mm, P = 0.024). No positional differences were observed in psoas lateralization, CSA, longitudinal diameter or transverse diameter. When prone, IVC was found to significantly migrate anteriorly (7.24 mm prone vs 2.56 mm supine, P < 0.001). However, decreased anterior displacement was demonstrated at more caudal levels (ρ: -0.38, P = 0.004). There were no significant differences in the anatomic locations of the aorta and iliac vasculature. Average segmental lordosis significantly increased when prone (14.62° prone vs 11.39° supine, P < 0.001). <h3>CONCLUSIONS</h3> This study suggests that anatomical relationships between the vertebral column, psoas muscle, and great vessels may change in the prone position and differ intraoperatively from supine imaging. Relative to the vertebral body, the psoas and IVC demonstrated substantial anterior mobility when prone– particularly at more cephalad levels– thereby promoting a safer intraoperative environment. Additionally, prone position enhanced segmental lordosis may be critical to optimizing sagittal restoration pre-discectomy. Further clinical investigations are necessary to corroborate these findings in a prone lateral cohort. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.