Background: The proximity of the posterior interosseous nerve (PIN) to the bicipital tuberosity is clinically important in the increasingly popular anterior single-incision technique for distal biceps tendon repair. Maximal forearm supination is recommended during tendon reinsertion from the anterior approach to ensure the maximum protective distance of the PIN from the bicipital tuberosity. Purpose: To compare the location of the PIN on magnetic resonance imaging (MRI) relative to bicortical drill pin instrumentation for suspensory button fixation via the anterior single-incision approach in varying positions of forearm rotation. Study Design: Descriptive laboratory study. Methods: Axial, non–fat suppressed, T1-weighted MRI scans of the elbow were obtained in positions of maximal supination, neutral, and maximal pronation in 13 skeletally mature individuals. Distances were measured from the PIN to (1) the simulated path of an entering guidewire (GWE-PIN) and (2) the cortical starting point of the guidewire on the bicipital tuberosity (CSP-PIN) achievable from the single-incision approach. To radiographically define the location of the nerve relative to constant landmarks, measurements were also made from the PIN to (3) the prominent-most point on the bicipital tuberosity (BTP-PIN) and (4) a perpendicular plane trajectory from the bicipital tuberosity exiting the opposing radial cortex (PPT-PIN). All measurements were subsequently compared between positions of pronation, neutral, and supination. In supination only, BTP-PIN and PPT-PIN measurements were made and compared at 3 sequential axial levels to evaluate the longitudinal course of the nerve relative to the bicipital tuberosity. Results: Of the 13 study participants, mean age was 38.77 years, and mean body mass index was 25.58. Five participants were female, and 5 left and 8 right elbow MRI scans were reviewed. The GWE-PIN was significantly greater in supination (mean ± SD, 16.01 ± 2.9 mm) compared with pronation (13.66 ± 2.5 mm) ( P < .005). The mean CSP-PIN was significantly greater in supination (16.20 ± 2.8 mm) compared with pronation (14.18 ± 2.4 mm) ( P < .013).The mean PPT-PIN was significantly greater in supination (9.00 ± 3.0 mm) compared with both pronation (1.96 ± 1.2 mm; P < .001) and neutral (4.73 ± 2.6 mm; P < .001). The mean BTP-PIN was 20.54 ± 3.0, 20.81 ± 2.7, and 20.35 ± 2.9 mm in pronation, neutral, and supination, respectively, which did not significantly differ between positions. In supination, the proximal, midportion, and distal measurements of BTP-PIN did not significantly differ. The proximal PPT-PIN distance (9.08 ± 2.9 mm) was significantly greater than midportion PPT-PIN (5.85 ± 2.4 mm; P < .001) and distal BTP-PIN (2.27 ± 1.8 mm; P < .001). Conclusion: This MRI study supports existing evidence that supination protects the PIN from the entering guidewire instrumentation during anterior, single-incision biceps tendon repair using cortical button fixation. The distances between the entering guidewire trajectory and PIN show that guidewire-inflicted injury to the nerve is unlikely during the anterior single-incision approach. Clinical Relevance: When a safe technique is used, PIN injuries during anterior repair are likely the result of aberrant retractor placement, and we recommend against the use of retractors deep to the radial neck. Guidewire placement as close as possible to the anatomic footprint of the biceps tendon is safe from the anterior approach. MRI evaluation confirms that ulnar and proximal guidewire trajectory is the safest technique when using single-incision bicortical suspensory button fixation.