Altered metabolism is a hallmark of cancer. In addition to glucose, glutamine is an important nutrient for cellular growth and proliferation. Noninvasive imaging via PET may help facilitate precision treatment of cancer through patient selection and monitoring of treatment response. l-[5-C]-glutamine (C-glutamine) is a PET tracer designed to study glutamine uptake and metabolism. The aim of this first-in-human study was to evaluate the radiologic safety and biodistribution of C-glutamine for oncologic PET imaging. Nine patients with confirmed metastatic colorectal cancer underwent PET/CT imaging. Patients received 337.97 ± 44.08 MBq of C-glutamine. Dynamic PET acquisitions that were centered over the abdomen or thorax were initiated simultaneously with intravenous tracer administration. After the dynamic acquisition, a whole-body PET/CT scan was acquired. Volume-of-interest analyses were performed to obtain estimates of organ-based absorbed doses of radiation. C-glutamine was well tolerated in all patients, with no observed safety concerns. The organs with the highest radiation exposure included the bladder, pancreas, and liver. The estimated effective dose was 4.46E-03 ± 7.67E-04 mSv/MBq. Accumulation of C-glutamine was elevated and visualized in lung, brain, bone, and liver metastases, suggesting utility for cancer imaging. PET using C-glutamine appears safe for human use and allows noninvasive visualization of metastatic colon cancer lesions in multiple organs. Further studies are needed to elucidate its potential for other cancers and for monitoring response to treatment.