We have previously shown that microgravity and simulated microgravity induces an increase in human and rat aortic stiffness. We attempted to elucidate the mechanism(s) responsible for this increase in stiffness. We hypothesize that an alteration in vessel wall collagen or elastin content or in extracellular matrix crosslinking (ECM) either individually or in a combination is responsible for the increased vessel stiffness. Rats underwent hindlimb unweighting (HLU) for a period of 7 days to simulate microgravity. The contribution of ECM crosslinking to the vessel wall stiffness was evaluated by measuring aortic pulse wave velocity following inhibition of the crosslinking enzymes lysyl oxidase (LOX) and transglutaminase (tTG) and the non-enzymatic advanced glycation endproduct (AGE) crosslinking pathway during HLU. Aortic collagen and elastin content was quantified using established, colorimetric assays. Collagen subtype composition was determined via immunofluorescent staining. The increase in aortic pulse wave velocity after HLU was significantly attenuated in the LOX and tTG inhibition groups, compared to saline (1.13 ± 0.11 m/s vs. 3.00 ± 0.15 m/s, LOX vs. saline, p<0.001; 1.16 ± 0.25 m/s vs. 3.00 ± 0.15 m/s, tTG vs. saline, p<0.001). Hydroxyproline content, a measure of collagen content, was increased in all groups after HLU (2.01±0.62% dry wt. vs. 3.69±0.68% dry wt., non-HLU vs. HLU, p=0.009). Collagen subtype composition and aortic elastin content were not altered by HLU. Together, these data indicate that HLU induced increases in aortic stiffness is due to both increased aortic collagen content and enzyme crosslinking activity.