Seipin regulates lipid homeostasis by preventing lipid droplet (LD) formation in non-adipocytes but promoting it in developing adipocytes. Here, we report that seipin interacts with 14-3-3β through its N- and C-termini. Expression of 14-3-3β is upregulated during adipogenesis, and its deletion results in defective adipogenesis without affecting key adipogenic transcription factors. We further identified the actin-severing protein cofilin-1 as an interacting partner to 14-3-3β. Cofilin-1 was spatiotemporally recruited by 14-3-3β in the cytoplasm during adipocyte differentiation. Extensive actin cytoskeleton remodelling, from stress fibres to cortical structures, was apparent during adipogenesis, but not under lipogenic conditions, indicating that actin cytoskeleton remodelling is only required for adipocyte development. Similar to seipin and 14-3-3β, cofilin-1 knockdown led to impaired adipocyte development. At the cellular level, differentiated cells with knockdown of cofilin-1, 14-3-3β or seipin continued to maintain relatively intact stress fibres, in contrast to cortical actin structure in control cells. Finally, 3T3-L1 cells expressing a severing-resistant actin mutant exhibited impaired adipogenesis. We propose that seipin regulates adipogenesis by recruiting cofilin-1 to remodel actin cytoskeleton through the 14-3-3β protein.