Women use more fat during endurance exercise as evidenced by a lower respiratory exchange ratio (RER). The contribution of intramyocellular lipid (IMCL) to lipid oxidation during endurance exercise is controversial, and studies investigating sex differences in IMCL utilization have found conflicting results. We determined the effect of sex on net IMCL use during an endurance exercise bout using an ultrastructural evaluation. Men ( n = 17) and women ( n = 19) completed 90-min cycling at 63% V̇o2peak. Biopsies were taken before and after exercise and fixed for electron microscopy to determine IMCL size, # IMCL/area, IMCL area density, and the % IMCL touching mitochondria. Women had a lower RER and carbohydrate oxidation rate and a higher lipid oxidation rate during exercise ( P < 0.05), compared with men. Women had a higher # IMCL/area and IMCL area density ( P < 0.05), compared with men. Women, but not men, had a higher % IMCL touching mitochondria postexercise ( P = 0.03). Exercise decreased IMCL area density ( P = 0.01), due to a decrease in the # IMCL/area ( P = 0.02). There was no sex difference in IMCL size or net use. In conclusion, women have higher IMCL area density compared with men, due to an increased # IMCL and not an increased IMCL size, as well as an increased % IMCL touching mitochondria postexercise. Endurance exercise resulted in a net decrease in IMCL density due to decreased number of IMCL, not decreased IMCL size, in both sexes.