Glioblastoma is the most aggressive primary brain tumor in adults with a median survival of less than 2 years. Radiolabeled amino acids like O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) have tremendous value in the diagnosis and treatment monitoring of gliomas with positron emission tomography (PET). However, the existence of [18F]FET PET negativity in 10–30% of patients with glioma underscores the need for alternative radiotracers. We recently reported that 6-[18F]fluoronicotinic acid ([18F]FNA) PET can clearly delineate intracranial human glioblastoma xenograft in a mouse model and that monocarboxylate transporters 1 and 2 (MCT1/2) mediate tumor uptake. This study evaluated the potential clinical utility of [18F]FNA by comparing its PET imaging performance with the current clinical amino acid radiotracers, [18F]FET and [11C]methionine ([11C]MET), as benchmarks. Orthotopic human glioblastoma xenograft models were prepared in mice for radiotracer comparisons and MCT blocking studies. Dynamic PET imaging, ex vivo biodistribution, brain tissue autoradiography, and histological staining were performed. In silico docking was also performed to analyze the interactions between MCT1 and FNA. The tumor was distinctly visualized on PET with [18F]FNA, whereas [18F]FET did not provide discernible tumor images in the same mice. Despite the negative [18F]FET PET results, the tumor was [11C]MET PET positive. In vivo blocking experiments indicated that MCT1, MCT2, and MCT4 are important transporters but may contribute in unequal measures to [18F]FNA tissue uptake. In vivo blocking with AZD3965, a dual inhibitor of MCT1 and MCT2, reduced [18F]FNA tumor uptake by 68–77%, while blocking MCT4 reduced uptake by 23%. In silico analysis supports ligand–protein interaction between FNA and MCT1. We were not able to visualize the human glioblastoma xenografts in mice by [18F]FET PET, but tumor uptake was detected with [18F]FNA and [11C]MET PET. MCT1, MCT2, and MCT4 have significant roles in [18F]FNA uptake in glioblastoma and other tissues. [18F]FNA could be an alternative approach for clinical PET imaging of glioblastoma based on a biological mechanism completely different from current clinical approaches.