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Genetic variation at the (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic -targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher expression than rs67785913 CT cells. Perturbed expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of , which originates in the maintenance of mitochondrial function, providing potential explanations for why genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.