Introduction: Pancreatic adenocarcinoma holds one of the worst prognosis of all known malignancies. Local treatment options for patients with unresectable pancreatic cancer may increase tumor control and benefit patient survival. This study aimed to test the feasibility of a possible new treatment method utilizing intratumoral holmium microsphere injections. Method: In this study, non-radioactive holmium-165 poly(L-lactic acid) microspheres (165Ho-PLLA-MS, HoMS) were injected in pancreatic adenocarcinoma in an ex-vivo setting. Patients signed informed consent to donate their resected pancreas with tumor to this study after conventional pancreatectomy. HoMS were brought in a suspension of Pluronic and Saline (5 mg HoMS per milliliter suspension) and was homogenized using an intratumoral injection device for HoMS. Pre- and post-injection MRI and CT were analyzed for holmium distribution. Visual holmium artefacts on MRI were manually segmented for a volume estimation. Results: Three tissue samples of 9.4 ml, 5.6 ml, and 11.2 ml were injected with 4.95 ml, 2.3 ml, and 1.8 ml HoMS suspension, respectively. Pre-injection MRI was used for treatment planning. Two tissue samples were injected using the pre-injection MRI and tumor palpation. In the last-sample additional ultrasound-guidance was performed. Injections were performed with minor leakage or needle obstruction. MRI segmentation showed fractions of the holmium artefacts visual in or near the tumor increased per sample with 26%, 43% and 68%, for sample 1, 2 and 3, respectively. Holmium quantification was performed on sample 3 and gave a simulated mean tumor dose 6 Gy and a mean dose in the largest deposits of 33 Gy. Small deposits were most likely excluded from quantification because of a low resolution. HoMS were not visible on CT. This was most likely caused by automated scanning parameters and a low HoMS concentration. Conclusion: Preliminary results show that intratumoral injection of HoMS in pancreatic adenocarcinoma is feasible in an ex-vivo setting. Improvements regarding microsphere concentration, imaging and quantification may be investigated during the final three patients in this trial. Considering the already reached distribution and injection control, intratumoral holmium microsphere injection may soon benefit patients with unresectable pancreatic cancer.