Matrix metalloproteinase-9 (MMP-9) is involved in tissue remodeling or neural plasticity in various clinical states (e.g. inflammation, neuropathic pain). We focused on the effect of MMP-9 on development of morphine tolerance after repeated morphine treatment. To develop morphine tolerance, mice were given morphine (10mg/kg; s.c.) once daily for 5days. The antinociceptive effect of morphine was measured by the tail flick method. Development of morphine tolerance was significantly inhibited by daily treatment of the non-specific MMP inhibitor GM6001 (5μg/mouse, i.c.v.). A MMP-9 inhibitor (5μg/mouse, i.c.v.) partially, yet significantly, inhibited the development of morphine tolerance. Intrathecal treatment of a MMP-9 inhibitor did not affect morphine tolerance. In MMP-9 (−/−) mice, the development of morphine tolerance was partially, yet significantly, inhibited compared with wild-type mice. MMP-9 protein expression levels in the midbrain gradually increased 12h to 24h after morphine treatment on day 1, but were unchanged on days 3–5. In the spinal cord, MMP-9 protein expression levels were unchanged. In gelatin zymography analyses, MMP-9 activity in the midbrain gradually increased 12 to 24h after morphine treatment. Increment in MMP-9 activity in the midbrain was also observed on days 3–5. Our findings suggest that persistent MMP-9 activation observed after the transient increment in MMP-9 expression from the early phase of morphine treatment may contribute to the development of morphine tolerance.