A high level of EpCAM overexpression in lung cancer makes this protein a promising target for targeted therapy. Radionuclide visualization of EpCAM expression would facilitate the selection of patients potentially benefiting from such treatment. Single-photon computed tomography (SPECT) using Tc-labeled engineered scaffold protein DARPin Ec1 has shown its effectiveness in imaging tumors with overexpression of EpCAM in preclinical studies, providing high contrast just a few hours after injection. This first-in-human study aimed to evaluate the safety and distribution of [Tc]Tc(CO)-(HE)-Ec1 in patients with primary lung cancer. Twelve lung cancer patients were injected with 300.7 ± 103.2 MBq of [Tc]Tc(CO)-(HE)-Ec1. Whole-body planar imaging (at 2, 4, 6 and 24 h after injection) and SPECT/CT of the lung (at 2, 4, and 6 h) were performed. The patients' vital signs and possible side effects were monitored up to 7 days after injection. The patients tolerated the injection of [Tc]Tc(CO)-(HE)-Ec1 well, and their somatic condition remained normal during the entire follow-up period. There were no abnormalities in blood and urine tests after injection of [Tc]Tc(CO)-(HE)-Ec1. The highest absorbed doses were in the kidneys, liver, pancreas, thyroid, gallbladder wall, and adrenals. There was also a relatively high accumulation of [Tc]Tc(CO)-(HE)-Ec1 in the small and large intestines, pancreas and thyroid. According to the SPECT/CT, accumulation of [Tc]Tc(CO)-(HE)-Ec1 in the lung tumor was found in all patients included in the study. Intensive accumulation of [Tc]Tc(CO)-(HE)-Ec1 was also noted in regional metastases. [Tc]Tc(CO)-(HE)-Ec1 can potentially be considered a diagnostic tracer for imaging EpCAM expression in lung cancer patients and other tumors with overexpression of EpCAM.