Obesity is characterized by aberrant fat accumulation. However, the intracellular signaling pathway that senses dietary fat and leads to fat storage remains elusive. Here, we have observed that the levels of histone deacetylase 6 (HDAC6) and the related family member HDAC10 are markedly reduced in adipose tissues of obese animals and humans. Mice with adipocyte-specific depletion of Hdac6 exhibited increased fat accumulation and reduced insulin sensitivity. In normal adipocytes, we found that reversal of P300/CBP-associated factor–induced (PCAF-induced) acetylation at K56 on cell death-inducing DFFA-like effector C (CIDEC, also known as FSP27) critically regulated lipid droplet fusion and lipid storage. Importantly, HDAC6 deacetylates CIDEC, leading to destabilization and reduced lipid droplet fusion. Accordingly, we observed elevated levels of CIDEC and its acetylated form in HDAC-deficient adipocytes as well as the adipose tissue of obese animals and humans. Fatty acids (FAs) prevented CIDEC deacetylation by promoting the dissociation of CIDEC from HDAC6, which resulted in increased association of CIDEC with PCAF on the endoplasmic reticulum. Control of CIDEC acetylation required the conversion of FAs to triacylglycerols. Thus, we have revealed a signaling axis that is involved in the coordination of nutrient availability, protein acetylation, and cellular lipid metabolic responses.