Cardiovascular disease (CVD) is the leading cause of death among women worldwide, and risk for developing CVD increases postmenopause. Consumption of trans-fatty acids (tFA) has been positively associated with CVD incidence and mortality. The current study was designed to assess the effects of diets high in industrially produced (IP)-tFA, from partially hydrogenated vegetable oils (PHVO), and ruminant-produced (RP)-tFA, from butter oil (BO), on risk factors for CVD. Thirty-two female Hartley guinea pigs, one-half of which were ovariectomized (OVX) to mimic the postmenopausal condition, were fed hypercholesterolemic diets containing 9% by weight PHVO or BO (n = 8/diet and ovariectomy) for 8 wk. The plasma and hepatic lipids did not differ between IP- and RP-tFA groups or between intact and OVX guinea pigs. The BO diet resulted in higher concentrations of plasma total and small HDL particle subclass concentrations than the PHVO diet regardless of ovariectomy status. The intact BO group had higher concentrations of large HDL particles than the intact PHVO group. HDL mean particle size tended to be larger (P = 0.07) in the PHVO groups compared with the BO groups regardless of ovariectomy status. There was a trend toward an interaction between diet and ovariectomy status for LDL mean particle size, which tended to be larger in OVX guinea pigs fed PHVO (P = 0.07). In summary, consumption of IP- and RP-tFA resulted in differential effects on HDL particle subclass profiles in female guinea pigs. The effect of tFA consumption and hormonal status on HDL particle subclass metabolism and the subsequent impact on CVD in females warrants further investigation.