Interventions affecting gastrointestinal (GI) physiology suggest that the GI tract plays an important role in modulating the uptake of ingested glucose by body tissues. We aimed at validating the use of positron emission tomography (PET) with oral 18FDG administration in mice, and to examine GI effects on glucose metabolism in adipose tissues, brain, heart, muscle, and liver, and interfering actions of oral lipid co-administration. We performed sequential whole-body PET studies in 3 groups of 10 mice, receiving i.p. glucose and 18FDG or oral glucose and 18FDG ± lipids, to measure tissue glucose uptake (GU) and GI transit, and compute the absorption lumped constant (LCa) as ratio of oral 18FDG-to-glucose incremental blood levels. GI and liver histology and circulating hormones were tested to generate explanatory hypothesis. Median LCa was 1.18, constant over time and not significantly affected by lipid co-ingestion. Compared to the i.p. route, the oral route (GI effect) resulted in lower GU rates in adipose tissues and brain, and a greater steatohepatitis score (+17%, p = 0.03). Lipid co-administration accelerated GI transit, in relation to the suppression in GIP, GLP1, glucagon, PP, and PYY (GI motility regulators), abolishing GI effects on subcutaneous fat GU. Duodenal crypt size, gastric wall 18FDG uptake, and macro-vesicular steatosis were inversely related to adipose tissue GU, and positively associated with liver GU. We conclude that 18FDG-PET is a suitable tool to examine the role of the GI tract on glucose transit, absorption, and bio-distribution. The GI effect consists in the suppression of glucose metabolism selectively in organs responsible for energy intake and storage, and is blunted by lipid ingestion. Modulation of gut and liver inflammation, as reflected by high GU, may be involved in the acute signalling of the energy status.