Adiponectin (ApN) is an adipokine, whose expression and plasma levels are inversely related to obesity and insulin-resistant states. Chronic repercussions of ApN treatment or overexpression on adiposity and body weight are still controversial. Here, we generated a transgenic mouse model allowing persistent and moderate overexpression of native full-length ApN targeted to white adipose tissue. Adipose mass and adipocyte size of Tg mice were reduced in spite of preserved calorie intake. This reduction resulted from increased energy expenditure and upregulation of uncoupling proteins, and from abrogation of the adipocyte differentiation program, as shown by the loss of a key lipogenic enzyme and of adipocyte markers. Adipose mass remodelling favours enhanced insulin sensitivity and improved lipid profile of Tg mice. Alteration of the adipocyte phenotype was likely to result from increased expression of the preadipocyte factor-1 (Pref-1) and from downregulation of the transcription factor, C/EBP that orchestrates adipocyte differentiation. We further found that recombinant ApN directly stimulated Pref-1 mRNA and attenuated C/EBP expression in cultured 3T3-F442A cells. Conversely, opposite changes in the expression of these genes were observed in white fat of ApN-deficient mice. Thus, besides enhanced energy expenditure, our work shows that impairment of adipocyte differentiation contributes to the anti-adiposity effect of ApN.