This paper is aimed to investigate the effect of rest-inserted loading on the mechanosensitivity of osteocytes. In the investigation, cultured MLO-Y4 osteocyte-like cells were strained on cyclic compressive force (CCF) by the self-made compressive loading device. Then we observed the effect of different rest periods-inserted loading (5 s, 15 s, 30 s, respectively) on the mechanosensitivity of osteocytes. We then determined the levels of secreted nitric oxide (NO) and prostaglandin E2 (PGE2) by Griess method and enzyme linked immunosorbent assay (ELISA), respectively. We then stained the cytoskeleton F-actin using immunofluorescence. We found that the expressions of NO and PGE2 in rest-inserted strained groups (>15 s) were significantly increased compared to those in the continuous strained group. And rest-inserted loading promoted the parallel alignment of stress fibers. It indicates that rest-inserted loading could promote the mechanosensitivity of osteocytes, and this might be related to the parallel alignment of stress fibers.