Intestinal fat absorption is known to be, overall, a highly efficient process, but much less is known about the efficiency with which individual dietary fatty acids (FA) are absorbed by the adult small intestine. We therefore measured the absorption efficiency of the major dietary FA using sucrose polybehenate (SPB) as a non-absorbable marker, and analyzed how it is modulated by acyl chain physicochemical properties and polymorphisms of proteins involved in chylomicron assembly. Dietary FA absorption efficiency was measured in 44 healthy subjects fed a standard diet containing 35% fat and 5% SPB. FA and behenic acid (BA) were measured in homogenized diets and stool samples by GC-mass spectroscopy, and coefficients of absorption for each FA were calculated as 1-[(FA/BA)_feces/(FA/BA)_diet]. Absorption coefficients for saturated FA decreased with increasing chain length and hydrophobicity (mean ± SE), and ranged from 0.95 ± 0.02 for myristate (14:0), 0.80 ± 0.03 for stearate (18:0), to 0.26 ± 0.02 for arachidate (20:0). Absorption coefficients for unsaturated FA increased with increasing desaturation from 0.79 ± 0.03 for elaidic acid (18:1t), 0.96 ± 0.01 for linoleate (18:2), to near complete absorption for eicosapentaenoic (20:5) and docosahexaenoic (22:6) acids. Of several common genetic polymorphisms in key proteins involved in the chylomicron assembly pathway, only the intestinal fatty acid binding protein-2 A54T allele (rs1799883) had any impact on FA absorption. We conclude that acyl chain length, saturation, and hydrophobicity are the major determinants of the efficiency with which dietary FA are absorbed by the adult small intestine.