In the present study, we isolated preadipocytes from the adipose tissue of Peking duck and subsequently cultured them in vitro. Cell counting kit-8 assay was employed to establish the growth curve of duck primary preadipocytes. Meanwhile, after the cells reaching full confluency, they were induced to differentiate into mature adipocytes by the addition of a cocktail containing dexamethasone, insulin, 3-isobutyl-1-methylxanthine, and oleic acid for 8 days. Successful differentiation was demonstrated by the development of lipid droplets and the expression of key marker genes including peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer binding protein-α (CEBP/α) and adipocyte fatty acid-binding protein (FABP4). Our results showed that duck primary preadipocytes began to adhere 12 h after seeding as short spindle shapes or litter triangles, which grew quickly 3 days post attachment and maintained stable after day 7. After 8 days the preadipocytes were induced to differentiate into mature adipocytes, which were stained red by oil red O. Additionally, it showed that during preadipocyte differentiation PPARγ mRNA was highly expressed at day 3, while CEBP/α and FABP4 mRNA peaked at day 5 and 8, respectively. These results indicate that we have successfully isolated and cultured Peking duck preadipocytes and successfully induced them to differentiate into mature adipocytes. This work could lay a foundation for further research into waterfowl adipogenesis.