Macrophage density and phenotype are key components of the innate immune response and have been implicated in the outcomes and progression of cancer and autoimmune diseases. The ability to quantify and perform temporospatial analysis of macrophages could enable more specific therapeutic interventions. Macrin, a 17-nm nanoparticle, has shown high selectivity for macrophage accumulation in preclinical models of disease. The purpose of this first-in-human study was to evaluate the biodistribution, dosimetry, and safety of 64Cu-labeled Macrin and to explore its potential for imaging inflammatory and malignant conditions. Methods: This study (NCT04843891) enrolled 10 human subjects (7 healthy volunteers and 3 patient subjects) who received a single administration of 64Cu-Macrin. The mean administered activity was approximately 444 MBq. Serial PET/CT scans were performed to determine the whole-body biodistribution and to calculate radiation dosimetry. Blood samples were collected at multiple time points to assess the pharmacokinetic profile. Participants were monitored for any adverse events or pharmacologic effects. Results: No adverse or clinically detectable pharmacologic effects were observed in any of the 10 participants, establishing the safety of the imaging agent. The blood clearance of 64Cu-Macrin was biphasic, with a fast component half-life of 1.3 h and a slow component of 22.3 h. In participants with diagnosed cancer and sarcoidosis, notable accumulation of 64Cu-Macrin was observed at sites of active disease. Conclusion: The results of this first-in-human study support the safety and favorable pharmacokinetic profile of 64Cu-Macrin PET imaging. The tracer successfully accumulated in macrophage-rich tissues, such as sites of cancer and sarcoidosis, suggesting that this approach could be a valuable tool for patient stratification and therapy response assessment, particularly in the development of macrophage-targeted therapeutics.