STK16 is a ubiquitously expressed, myristoylated, and palmitoylated serine/threonine protein kinase with underexplored functions. Recently, it was shown to be involved in cell division but the mechanism remains unclear. Here we found that human STK16 localizes to the Golgi complex throughout the cell cycle and plays important roles in Golgi structure regulation. STK16 knockdown or kinase inhibition disrupts actin polymers and causes fragmented Golgi in cells. In vitro assays show that STK16 directly binds to actin and regulates actin dynamics in a concentration- and kinase activity-dependent way. In addition, STK16 knockdown or kinase inhibition not only delays mitotic entry and prolongs mitosis, but also causes prometaphase and cytokinesis arrest. Therefore, we revealed STK16 as a novel actin binding protein that resides in the Golgi, which regulates actin dynamics to control Golgi structure and participate in cell cycle progression.