In early-stage breast cancer, the primary treatment option for most women is breast-conserving surgery (BCS). There is a clear need for more accurate techniques to assess resection margins intraoperatively, because on average 20% of patients require further surgery to achieve clear margins. Cerenkov luminescence imaging (CLI) combines optical and molecular imaging by detecting light emitted by F-FDG. Its high-resolution and small size imaging equipment make CLI a promising technology for intraoperative margin assessment. A first-in-human study was conducted to evaluate the feasibility of F-FDG CLI for intraoperative assessment of tumor margins in BCS. Twenty-two patients with invasive breast cancer received F-FDG (5 MBq/kg) 45-60 min before surgery. Sentinel lymph node biopsy was performed using an increased Tc-nanocolloid activity of 150 MBq to facilitate nodal detection against the γ-probe background signal (cross-talk) from F-FDG. The cross-talk and Tc dose required was evaluated in 2 lead-in studies. Immediately after excision, specimens were imaged intraoperatively in an investigational CLI system. The first 10 patients were used to optimize the imaging protocol; the remaining 12 patients were included in the analysis dataset. Cerenkov luminescence images from incised BCS specimens were analyzed postoperatively by 2 surgeons blinded to the histopathology results, and mean radiance and margin distance were measured. The agreement between margin distance on CLI and histopathology was assessed. Radiation doses to staff were measured. Ten of the 12 patients had an elevated tumor radiance on CLI. Mean radiance and tumor-to-background ratio were 560 ± 160 photons/s/cm/sr and 2.41 ± 0.54, respectively. All 15 assessable margins were clear on CLI and histopathology. The agreement in margin distance and interrater agreement was good (κ = 0.81 and 0.912, respectively). Sentinel lymph nodes were successfully detected in all patients. The radiation dose to staff was low; surgeons received a mean dose of 34 ± 15 μSv per procedure. Intraoperative F-FDG CLI is a promising, low-risk technique for intraoperative assessment of tumor margins in BCS. A randomized controlled trial will evaluate the impact of this technique on reexcision rates.