Objective To investigate the effects of cyclic stretching strain on the cytoskeleton of human periodontal ligament cells (HPDLCs). Methods HPDLCs were cultured in vitro, and were cyclicly stretched by the strain of 1%, 10% and 20% for 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h and 24 h, respectively. The morphology of cytoskeleton of HPDLCs was observed by confocal laser scanning microscopy, and figures were collected for analysis with ImagePro Plus 4.5.0.29 software. The area of HPDLCs, ratio of length to width and  integrated fluorescence intensity of cytoskeleton protein F-actin were measured. Results Confocal laser scanning microscopy revealed that with the increase of stretching strain and prolongation of action, HPDLCs gradually arranged in parallel and exhibited paliform, and the direction of arrangement was vertical to that of stretching strain, with prolonged cell body. Figure analysis demonstrated that the area of HPDLCs, ratio of length to width and expression of F-actin changed accordingly after cyclic stretching strain load. Conclusion Cyclic stretching strain may cause changes of cytoskeleton of HPDLCs, which may be related to the level of stretching strain and duration of action.