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ABSTRACT To explore the functions of Txnip and its mechanism in adipocyte differentiation, the preadipocytes were isolated from subcutaneous adipose tissue of three-day-old piglets and induced into adipogenic differentiation. The expression of Txnip and ChREBP was silenced, and the Txnip overexpression was achieved in the cells with transfection of the recombinant lentivirus strategies. Txnip silencing promoted the differentiation of porcine preadipocytes and PPARγ expression, and a PPARγ inhibitor reduced this facilitation. Instead, Txnip overexpression exerted a suppressive effect on the cell differentiation and PPARγ expression, and the PPARγ agonist offset this inhibition. High glucose stimulated the preadipocyte differentiation and expressions of ChREBP and Txnip. In contrast, Txnip expression was reduced by ChREBP silencing, suggesting glucose-regulated Txnip expression through the mediation of ChREBP. Moreover, the expressions of ChREBP and Glut4 induced by high glucose and glucose uptake of the cells were reduced by Txnip-overexpression, but increased by Txnip silencing, while these changes of Txnip did not alter their expressions under low glucose. Collectively, Txnip could be an inhibitor of porcine preadipocyte differentiation, which attenuated the adipogenesis through the negative feedback regulation on PPARγ. Txnip impaired the induction of glucose on the preadipocyte differentiation through decreasing Glut4 expression and glucose uptake and subsequent decrease of the expression and transcriptional activity of ChREBP.