Premature Ovarian Insufficiency (POI) is a heterogeneous condition characterized by accelerated follicular atresia and ovarian cessation before the age of 40. Mitochondria, the energy-producing organelles of the cell, play a vital role in folliculogenesis by supplying ATP through oxidative phosphorylation and the mitochondrial fatty acid shuttle (MFAS). The MFAS pathway involves key enzymes such as CPT1, CPT2, CACT, and OCTN2, which facilitate the transport of long-chain fatty acids into the mitochondrial matrix for β-oxidation. Despite these enzymes playing a critical role in cellular energy metabolism, their role in POI remains largely unexplored. This study aimed to identify novel mitochondrial variants associated with POI through exome sequencing in affected women and validate the findings using mouse models. Whole-exome sequencing of women with idiopathic POI, followed by in-silico analysis and protein modeling, identified a novel variant in OCTN2 (SLC25A5) and variants of uncertain significance in MFAS-related genes CPT1A, CPT1B, and CPT2. Additional novel pathogenic variants were found in ARHGEF28 and AOX1, along with likely pathogenic variants in mitochondrial genes MT-ND2, MT-ATP6, MT-CYB, MT-ND4, MT-ND5, and MT-CO3. Several known POI-associated pathogenic variants and VOUS in mitochondrial and cell-cycle-related genes were also observed. To evaluate the functional significance of MFAS disruption, C57BL/6 adolescent mice (3–5 weeks) were treated with inhibitors including Etomoxir, Omeprazole, L-Amino Carnitine, Etoposide, and Mildronate, with cyclophosphamide as a positive control. Treated mice exhibited reduced body and ovarian weight, while histological analysis revealed decreased follicle numbers, increased follicular atresia, and disrupted ovarian architecture—findings comparable to cyclophosphamide-treated mice. Additionally, the liver, kidney, and spleen displayed altered morphology, highlighting systemic effects of MFAS inhibition. Serum FSH levels were elevated in treated groups, confirming a POI-like hormonal profile. These findings underscore mitochondrial dysfunction and MFAS impairment in POI, highlighting fatty acid oxidation as a key regulator of ovarian function.