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To prove that, the metabolic and molecular mechanisms responsible for HAdV31\u2010induced adipogenesis were examined. 3T3L1 cells (mouse embryonic fibroblast, adipose like cell line) were used as a surrogate model to analyze an increased proliferation, differentiation, and maturation of preadipocytes infected with human adenovirus. An expression of E4orf1, C\/EBP\u2010\u03b2, PPAR\u2010\u03b3, GAPDH, aP2, LEP, and fatty acid synthase genes, intracellular lipid accumulation as well as cytokine release from the fat cells were assessed. Data showed that HAdV31 increased an expression of C\/EBP\u2010\u03b2 and PPAR\u2010\u03b3 genes leading to an enhanced differentiation of preadipocytes into fat cells. Besides, overexpression of GAPDH and fatty acid synthase, and decreased expression of leptin caused an increased accumulation of intracellular lipids. Secretion of TNF\u2010\u03b1 and IL\u20106 from HAdV31\u2010infected cells was strongly decreased, leading to unlimited virus replication. The results obtained from this study provided the evidences that HAdV31, likewise previously documented HAdV36, is a subsequent human adenovirus affecting the differentiation and lipid accumulation of 3T3L1 cells. <jats:bold><jats:italic>J. Med. 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