Purpose To investigate the sustained-attention and memory-enhancing neural correlates of the oral administration of methylene blue in the healthy human brain. Materials and Methods The institutional review board approved this prospective, HIPAA-compliant, randomized, double-blinded, placebo-controlled clinical trial, and all patients provided informed consent. Twenty-six subjects (age range, 22-62 years) were enrolled. Functional magnetic resonance (MR) imaging was performed with a psychomotor vigilance task (sustained attention) and delayed match-to-sample tasks (short-term memory) before and 1 hour after administration of low-dose methylene blue or a placebo. Cerebrovascular reactivity effects were also measured with the carbon dioxide challenge, in which a 2 × 2 repeated-measures analysis of variance was performed with a drug (methylene blue vs placebo) and time (before vs after administration of the drug) as factors to assess drug × time between group interactions. Multiple comparison correction was applied, with cluster-corrected P < .05 indicating a significant difference. Results Administration of methylene blue increased response in the bilateral insular cortex during a psychomotor vigilance task (Z = 2.9-3.4, P = .01-.008) and functional MR imaging response during a short-term memory task involving the prefrontal, parietal, and occipital cortex (Z = 2.9-4.2, P = .03-.0003). Methylene blue was also associated with a 7% increase in correct responses during memory retrieval (P = .01). Conclusion Low-dose methylene blue can increase functional MR imaging activity during sustained attention and short-term memory tasks and enhance memory retrieval. ^© RSNA, 2016 Online supplemental material is available for this article.