Some of the key steps in adenomyosis (AM) are the migration and invasion of adenomyosis derived cells (AMDC); these processes require rearrangement of the cytoskeleton which Rho/ROCK signaling pathway is involved. The aim of the current study was to verify AMDC characterization and the effect of Trans-Cinnamaldehyde (TC) on the expression of the cell viability and cytotoxicity, cytoskeleton, invasion, migration, RhoA, RhoB, RhoC, ROCK1, ROCK2 in AMDC, which by immunofluorescence, CCK-8, immunofluorescence, cell wound scratch assay, transwell invasion assay and western blot analysis. Human endometrium tissues were obtained from AM patients who underwent total hysterectomy for AM in the Linyi Central Hospital from September 2019 to May 2021 with the first patient 41 years old and second one 45 years old. AMDC was characterized with strongly positive staining for Vimentin and almost negative for CK7, VWF, E-cadherin, SMA, and the patient samples were in secretory phases. IC50 = 28.93±0.44 μg/mL at 48 h with first patient, and IC50 = 27.67±0.72 μg/mL with second one. The final concentrations of TC (0, 6, 12μg/mL) were selected of the cell cytotoxicity. The T-text test group >0.05, so there was no difference between the two patients. These experiments were significantly downregulated and in dose-dependent fashion after treatment with TC. The data suggest that TC can inhibition the viability of cell, regulate cytoskeleton-induced invasion and migration may related to Rho/ROCK signaling pathway are involved in the pathophysiology of adenomyosis. Keyword: Cytoskeleton, Invasion, Migration, Rho/ROCK pathway, Trans-Cinnamaldehyde, Adenomyosis