Women utilize more fat during endurance exercise as evidenced by a lower RER. The contribution of 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 = 18) completed 90-min cycling @ 63% VO_2peak. 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 CHO oxidation rate and a higher lipid oxidation rate during exercise (P < 0.05), as compared with men. Women had a higher # IMCL/area and IMLC area density (P < 0.05), as compared with men. Women, but not men, had a higher % IMCL touching mitochondria post exercise (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 as compared with men, due to an increased # IMCL and not an increased IMCL size, as well as an increased % IMCL touching mitochondria post exercise. Endurance exercise resulted in a net decrease in IMCL density due to decreased number of IMCL, not decreased IMCL size, in both sexes.