The peroxisome proliferator‐activated receptors (PPARs) bind and are activated by a variety of fatty acids and derivatives thereof. Agonist binding enhances PPAR‐mediated transactivation via release of corepressors and recruitment of coactivator complexes. Recently, we and others have reported that acyl‐CoA esters act as PPAR antagonists in vitro. Here, we show that the binding of the nonhydrolyzable acyl‐CoA analogue, S‐hexadecyl‐CoA, differentially affected conformation and coactivator recruitment of the individual PPAR subtypes. In protease protection assays, S‐hexadecyl CoA increased the sensitivity of PPARα and PPARδ towards chymotrypsin, whereas the action of chymotrypsin on PPARγ was only marginally affected, suggesting distinct subtype‐dependent differences in the effects of S‐hexadecyl‐CoA on conformation of the PPARs. In keeping with these findings, S‐hexadecyl‐CoA abrogated ligand‐induced recruitment of coactivators to PPARα and PPARδ, whereas coactivator recruitment to PPARγ was unaffected by S‐hexadecyl‐CoA.