Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, impacts cancer cell proliferation and plant stress responses. However, its role in plant cell dedifferentiation and callus formation is not well understood. This study explores the function of cytoplasmic G6PD isoforms in Arabidopsis pericycle cell reprogramming into callus by employing a suite of mutant analyses, qRT-PCR, and GC-MS. Our findings demonstrate that g6pd5/6 double mutants exhibit enhanced callus formation compared to wild-type and single mutants, implicating cytoplasmic G6PDs as negative regulators of callus development. The double mutant showed reduced NADPH levels and increased expression of very-long-chain fatty acid (VLCFA) biosynthesis genes and the VLCFA-downstream gene Aberrant Lateral Root Formation 4 (ALF4) on callus-inducing medium (CIM). Notably, VLCFA concentrations were decreased in g6pd5/6 mutants, and supplementation of VLCFA reduced callus area. Additionally, callus formation in the alf4/g6pd5/6 triple mutant aligned with wild-type, suggesting a redundant inhibitory function of G6PD5 and G6PD6 in the regulation of VLCFA accumulation and related signaling. Contrasting with their roles in cancer cell proliferation, our study unveils novel insights into the G6PD signaling pathway, highlighting its unique function in negatively regulating plant callus formation.