Background: Recently, the duodenum has garnered interest for its role in treating metabolic diseases, including type 2 diabetes (T2DM). Multiple sessions of external photobiomodulation (PBM) in previous animal studies suggested it resulted in improved hyperglycemia, glucose intolerance, and insulin resistance with a multifactorial mechanism of action, despite the target organ of PBM not being clearly proven. This study aimed to determine whether a single session of a duodenal light-emitting diode (LED) PBM may impact the T2DM treatment in an animal model. Methods: Goto–Kakizaki rats as T2DM models were subjected to PBM through duodenal lumen irradiation, sham procedure, or control in 1-week pilot (630 nm, 850 nm, or 630/850 nm) and 4-week follow-up (630 nm or 630/850 nm) studies. Oral glucose tolerance tests; serum glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide, and insulin levels; liver chemistry and histology; and gut microbiome in the PBM, sham control, and control groups were evaluated. Results: In the 1-week study, duodenal dual-wavelength (D, 630/850 nm) LED PBM showed improved glucose intolerance, alkaline phosphatase and cholesterol levels, and weight gain than other groups. The D-LED PBM group in the 4-week study also showed improved hyperglycemia and liver enzyme levels, with relatively preserved pancreatic islets and increased serum insulin and GLP-1 levels. Five genera (Bacteroides, Escherichia, Parabacteroides, Allobaculum, and Faecalibaculum) were significantly enriched 1 week after the D-LED PBM. Bacteroides acidifaciens significantly increased, while Lachnospiraceae significantly decreased after 1 week. Conclusion: A single session of D-LED PBM improved hyperglycemia and hepatic parameters through the change of serum insulin, insulin resistance, insulin expression in the pancreatic β-cells, and gut microbiome in T2DM animal models.