Background: Metabolic surgery has been increasingly recommended for obese diabetic patients, but questions remain as to its effectiveness for nonobese diabetic patients and its mechanism that leads to glucose homeostasis independently of weight loss. Methods: We evaluated the role of the Roux limb (RL) in Roux-en-Y gastric bypass (RYGB) surgery, which facilitates food outflow from the upper stomach pouch in nonobese diabetic rat models. Findings: Improvements in glucose metabolism were greater in the long-RL RYGB group than in the other operated RYGB groups or the Goto-Kakizaki (GK) controls. Moreover, diabetes remission by RYGB was found to be remarkably different from both the healthy and diabetic states in the analysis of transcriptome profiles of three rat models individually representing different physiological states. Furthermore, according to a functional analysis, RYGB surgery significantly affected one group of genes, i.e., the new-changed group, which was originally normal (not significantly different between GK and Wistar rats) but became abnormal in the RYGB group (significantly different between RYGB and Wistar rats), despite their diabetes remission. We further observed that novel of this new-changed group induced by RYGB played key roles in improved glycemic control to help rebalance intestinal glucose metabolism. In particular, an increase in newly synthesized cholesterol (CHOL) enriched by the new-changed group increased glucose consumption, which contributed to glucose homeostasis, as demonstrated by a biodistribution analysis using [14C]-labeled glucose. Interpretation: Our findings suggest glycometabolic rebalance by RYGB-induced dysfunctions in the new-changed group as a compensatory role contributes to diabetes remission. Additionally, we propose long-RL RYGB for diabetic patients without morbid obesity. Funding: National Key R&D Program of China (No. 2017YFA0505500), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB13040700), National Natural Science Foundation of China (Nos. 81471047, 31771476 and 81802354), and Shanghai Sailing Program (No. 18YF1420700). Author contributions Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: All experiments were in compliance with NIH guidelines and the Guide for the Care and Use of Laboratory Animals and were approved by the Chinese Academy of Science Institutional Animal Care and Use Committee.