The vascular smooth muscle cells (VSMCs) of the vessel wall are differentiated cells specialized in the contractile function required for the maintenance of vascular tone. However, VSMCs are not terminally differentiated and in response to local environmental factors can experience a phenotypic switch towards a synthetic phenotype, which leads to cell proliferation, migration and production of extracellular matrix components. Ion channels have been shown to participate in cell proliferation, as they can modulate the progression of the cells through the cell cycle. In this regard, it is known that in different cell types K+ channel expression varies with the cell cycle. Also, several reports indicate that the blockade of some K+ currents in VSMCs is antiproliferative. Recently, our group has demonstrated that the functional expression of Kv3.4 channels is related to proliferation of human uterine VSMCs. In this work, we sough to gain deeper insight on the relationship between the expression and function of Kv3.4 channels and the progression of cultured uterine VSMCs through the cell cycle. We have used selective cell cycle blockers to describe the expression pattern of Kv3.4 mRNA and protein and its functional contribution along the cell cycle in these cells. Besides, we have explored the effects of the blockade of Kv3.4 channels on the distribution of the cells in the different phases of the cell cycle. We found that Kv3.4 mRNA levels increased when cells enter in G1 phase, decline in G2/M and reach the lowest level in quiescent (G0) cells, but Kv3.4 protein levels and Kv3.4 currents remain elevated while cells are proliferating, being maximum during G2/M phase. On the other hand, Kv3.4 channels blockade leads to a decrease in the mRNA levels of D1, A2 and B1 cyclins, an increase in the proportion of cells in the G0/G1 phase, a decrease in the number of ki67-positive cells and a decrease in the proportion of cells incorporating BrdU. Altogether, our data indicates that the blockade of Kv3.4 channels induces a decrease in the number of cells entering the cell cycle, suggesting the existence of a close association between the functional expression of Kv3.4 channels and the proliferation rate of uterine VSMCs.