We evaluated the impact of gender differences in both the quantitative and qualitative features of HDL subspecies on cellular free cholesterol efflux through the scavenger receptor class B type I (SR-BI), ABCA1, and ABCG1 pathways. For that purpose, healthy subjects (30 men and 26 women) matched for age, body mass index, triglyceride, apolipoprotein A-I, and high density lipoprotein-cholesterol (HDL-C) levels were recruited. We observed a significant increase (+14%; P < 0.03) in the capacity of whole sera from women to mediate cellular free cholesterol efflux via the SR-BI-dependent pathway compared with sera from men. Such enhanced efflux capacity resulted from a significant increase in plasma levels of large cholesteryl ester-rich HDL2 particles (+20%; P < 0.04) as well as from an enhanced capacity (+14%; P < 0.03) of these particles to mediate cellular free cholesterol efflux via SR-BI. By contrast, plasma from men displayed an enhanced free cholesterol efflux capacity (+31%; P < 0.001) via the ABCA1 transporter pathway compared with that from women, which resulted from a 2.4-fold increase in the plasma level of prebeta particles (P < 0.008). Moreover, in women, SR-BI-mediated cellular free cholesterol efflux was significantly correlated with plasma HDL-C (r = 0.72, P < 0.0001), whereas this relationship was not observed in men. In conclusion, HDL-C level may not represent the absolute indicator of the efficiency of the initial step of the reverse cholesterol transport.