Objective To investigate the shape and F-actin of human periodontal ligament fibroblasts(HPDLF) under different tension stress in vitro so as to learn the stress-biological effects of HPDLF. Methods HPDLF were cultivated for 6 passages and observed morphologically and identified by immunocytochemistry to be positive anti-vimentin and negative anti-keratin. HPDLF were divided into four groups: control (without tension stress), static tension stress (5 kPa), dynamic tension stress group 1 (5 to 0 to 5 kPa at the frequency of 3/min) and dynamic tension stress group 2 (5 to 2.5 to 5 kPa at the frequency of 3/min). The cells of each group were observed at the different time points of 16, 24, 32 h. The projection areas and shapes of cells as well as the structure of F-actin were examined by laser scanning confocal microscope and immunity fluorescence technique. The relationship among tension stress, time, shape and the structure of F-actin of HPDLF was analysed. Results In the dynamic tension stress group 1 and 2, the shape and the arrangement of F-actin of some HPDLF underwent regular changes at 16, 24 h, but the changes appeared to be obvious at 32 h. In the static tension stress group, it was found the increase of interspace of HPDLF, but no obvious changes in the structure of F-actin. Conclusion Different tension stress patterns had different effects on the shape and F-actin of HPDLF. Especially in the dynamic tension stress group 2, it showed direct ratio between the cell projection areas, the average fluorescence density and different loading time.