The aim of this study is to observe the effect of different treatment time of millimeter wave (MMW) on chondrocyte apoptosis, caspase-3, caspase-8, and matrix metalloproteinase-13 (MMP-13) in rabbit knee osteoarthritis induced by anterior cruciate ligament transection (ACLT). Thirty-two New Zealand White rabbits were randomly assigned into 4 groups: millimeter wave treatment for 20-min group (MWT20); millimeter wave treatment for 40-min group (MWT40); model control group (MC) and normal control group (NC). All groups received anterior cruciate ligament transection in the right knee except NC group. Six weeks after transection, the MWT20 group and MWT40 group were given millimeter wave (MMW) at 37.5 GHz frequency, 8 mm wavelength, and 10 mW/cm(2) power for 20 and 40 min, respectively, for 10 days. Eight weeks after transection, all animals were killed. Modified Mankin Score was assessed for histological assessment. Chondrocytes apoptosis was tested by the TUNEL assessment, and the expressions of related proteins were tested by the immunohistochemistry observation and Western blot. The modified Mankin Score, the chondrocyte apoptosis, and the expression of caspase-3 and MMP-13 in MWT40 group were significantly lower than those in MC group. Only a decreasing trend of modified Mankin Score and caspase-3 and MMP-13 expression was found in MWT20 group. The caspase-8 expression of the treatment groups was lower than model control group and higher than normal control group, but no significant difference was found. This study revealed MWT40 had a better therapeutic benefit to osteoarthritis cartilage structure, decreased the apoptosis of chondrocyte, and caspase-3 and MMP-13 expression compared to MWT20. But only a decreasing trend of caspase-8 expression was found.