Cancer cells are dysregulated and addicted to continuous supply and metabolism of nutritional glucose and amino acids (e.g., arginine) to drive the synthesis of critical macromolecules for uncontrolled growth. Recent studies have revealed that genome-derived microRNA (miRNA or miR)-1291-5p (miR-1291-5p or miR-1291) may modulate the expression of argininosuccinate synthase (ASS1) and glucose transporter protein type 1 (GLUT1). We also developed a novel approach to produce recombinant miR-1291 agents for research, which are distinguished from conventional chemo-engineered miRNA mimics. Herein, we firstly demonstrated that bioengineered miR-1291 agent was selectively processed to high levels of target miR-1291-5p in human pancreatic cancer (PC) cells. After the suppression of ASS1 protein levels, miR-1291 perturbed arginine homeostasis and preferably sensitized ASS1-abundant L3.3 cells to arginine deprivation therapy. In addition, miR-1291 treatment reduced the protein levels of GLUT1 in both AsPC-1 and PANC-1 cells, leading to a lower glucose uptake (deceased > 40%) and glycolysis capacity (reduced approximately 50%). As a result, miR-1291 largely improved cisplatin efficacy in the inhibition of PC cell viability. Our results demonstrated that miR-1291 was effective to sensitize PC cells to arginine deprivation treatment and chemotherapy through targeting ASS1- and GLUT1-mediated arginolysis and glycolysis, respectively, which may provide insights into understanding miRNA signaling underlying cancer cell metabolism and development of new strategies for the treatment of lethal PC. SIGNIFICANCE STATEMENT: Many anticancer drugs in clinical use and under investigation exert pharmacological effects or improve efficacy of coadministered medications by targeting cancer cell metabolism. Using new recombinant miR-1291 agent, we revealed that miR-1291 acts as a metabolism modulator in pancreatic carcinoma cells through the regulation of argininosuccinate synthase- and glucose transporter protein type 1-mediated arginolysis and glycolysis. Consequently, miR-1291 effectively enhanced the efficacy of arginine deprivation (pegylated arginine deiminase) and chemotherapy (cisplatin), offering new insights into development of rational combination therapies.