The gradual disorganization of collagen fibers in the stromal connective tissue of the uterine cervix is characteristic of progressive cervical softening during pregnancy. A lack of thrombospondin (TSP) 2 has been shown to be associated with altered collagen fibril morphology of connective-tissue-rich organs such as skin and tendon. The goal of this study was to determine the role of TSP2 in cervical softening by studying a TSP2-null mouse line. Creep testing showed that, in the nonpregnant animal and on Day 10 of pregnancy, there was no difference between the cervical extensibility of the wild-type and the TSP2-deficient mice. However, by Day 14 of pregnancy, the TSP2-null mice showed 4.5-fold increase in cervical extensibility, and by Day 18, a 6.1-fold increase, when compared with wild-type mice. A further indicator of compromised cervical integrity was that, on Days 14 and 18 of pregnancy, the cervix of TSP2-null mice broke rapidly under standard loading conditions that did not break the cervix of wild-type mice. Western blotting showed that TSP2 was expressed in the cervix of mice on Days 14 and 18 of pregnancy but not on Day 10 or in the nonpregnant animal. As determined by immunohistochemistry, the amount of matrix metalloproteinase 2 (MMP2) in the cervix of TSP2-null mice increased 11-fold on Day 14 of pregnancy and 19-fold on Day 18. Thus, TSP2-null mice provide an animal model to assist in the understanding of the molecular basis of spontaneous, premature softening of the uterine cervix.