Hypoxic pulmonary hypertension (HPH) leads to large pulmonary artery (PA) stiffening, which affects right ventricular afterload. We hypothesized that vascular collagen accumulation is the major cause of large PA stiffening in HPH. The hypothesis was tested with transgenic mice that synthesize collagen type I resistant to collagenase degradation (Col1a1^R/R) and wild-type controls. Animals were exposed to hypoxia for 10 days and then allowed to recover. Main PAs were harvested for mechanical and biochemical tests. Effects of smooth muscle cells (SMCs) were examined using the vasoactive agents U46619 and Y27632. Pressure-diameter testing showed that left PAs of both Col1a1^R/R and wild-type mice stiffened with hypoxia (p<0.01). Measurement of hydroxyproline biochemically confirmed that PA collagen content increased as well (p<0.01). After recovery, wild-type PAs were less stiff (p<0.01), whereas Col1a1^R/R PAs remained stiffened; biochemically, collagen content tended to decrease in both strains although less so in the Col1a1^R/R. Interestingly, after hypoxia, Col1a1^R/R PAs were less stiffened than those of wild-type mice. We speculate that differences in mean PA pressure during the 10 days of hypoxia between strains are responsible. Consistent with our hypothesis, Col1a1^R/R PAs also accumulated less collagen in response to hypoxia than wild-type mice. In the recovery group, stiffness and collagen content were comparable between strains. Quantitation for elastin showed no significant differences between groups. No significant differences between exposures and strains were observed in the mechanical effects of SMCs. The correlation between mechanical and biological properties suggests that collagen accumulation is critical to HPH-induced PA stiffening.