Previously, we reported that millimeter wave promoted the chondrocyte proliferation by pushing cell cycle progression. Activation of K(+) channels plays an essential role in the stimulating of extracellular matrix (ECM) synthesis and the cell proliferation in chondrocytes. While it is unclear if millimeter wave enhances ECM synthesis and proliferation of chondrocytes by regulating K(+) channel activity, we here investigated the effects of millimeter waves on ECM synthesis, chondrocyte proliferation and ion channels in the primary chondrocyte culture. We found that millimeter waves led to the increase of chondrocyte viability, the morphological changes of chondrocyte, and the F-actin distortion and remodeling. Ultrastructural analysis showed that treated chondrocytes contained an expansion of mitochondria and granular endoplasmic reticulum, and a high number of cytoplasmic vesicles in the cytoplasm compared to untreated cells, suggesting millimeter waves increased the energy metabolism and protein synthesis of chondrocytes. The analysis of differential ion channels' genes expression further showed an obvious increase of Kcne1, Kcnj3 and Kcnq2. To determine the role of voltage-gated K(+) channel in chondrocyte, we blocked the voltage-gated K(+) channel with 10 mM tetraethylammonium (TEA) and treated chondrocytes with millimeter waves. The results indicated that TEA significantly negated the promotion of millimeter waves for the ECM synthesis and chondrocyte proliferation. Our results support the hypothesis that millimeter waves promote the synthesis of ECM and the proliferation of chondrocyte by regulating the voltage-gated K(+) channel.