Secretory sphingomyelinase (sSMase) has been suggested to be involved in the development of cardiovascular diseases as well as other human pathologies. To deduce whether dietary fatty acid composition affects the circulating activity of this enzyme, we have compared its activity in serum from rats that had been given a diet containing either butter or a highly n‐6 polyunsaturated [grapeseed oil (GSO)] fat source for 14 wk. The results show that intake of GSO increases the activity of this ceramide‐producing enzyme by about 45%, when compared with intake of butter (387 ± 16 pmol/mL·h vs. 266 ± 15 pmol/mL·h; p <0.001). Furthermore, there was a strong negative correlation between sSMase activity and n‐3 PUFA concentration in serum (p <0.001). Despite the substantial increase in activity, there was no difference in either the circulating substrate (sphingomyelin) or product (ceramide) in the serum. However, since the sSMase activity in the endothelial wall has been implicated to be involved in both atherogenesis and thrombosis, these findings are of interest in the interpretation of dietary fatty acid effects on cardiovascular health.