The mechanisms behind the anti-tumoral effects of cannabinoids by impacting the migratory activity of tumor cells are only partially understood. Previous studies demonstrated that cannabinoids altered the organization of the actin cytoskeleton in various cell types. As actin is one of the main contributors to cell motility and is postulated to be linked to tumor invasion, we tested the following hypothesizes: 1) Can cannabinoids alter cell motility in a cannabinoid receptor dependent manner? 2) Are these alterations associated with reorganizations in the actin cytoskeleton? 3) If so, what are the underlying molecular mechanisms? Three different glioblastoma cell lines were treated with specific cannabinoid receptor 1 and 2 agonists and antagonists. Afterwards, we measured changes in cell motility using live cell imaging and alterations of the actin structure in fixed cells. Additionally, the protein amount of phosphorylated p44/42 mitogen-activated protein kinase (MAPK), focal adhesion kinases (FAK) and phosphorylated FAK (pFAK) over time were measured. Cannabinoids induced changes in cell motility, morphology and actin organization in a receptor and cell line dependent manner. No significant changes were observed in the analyzed signaling molecules. Cannabinoids can principally induce changes in the actin cytoskeleton and motility of glioblastoma cell lines. Additionally, single cell motility of glioblastoma is independent of their morphology. Furthermore, the observed effects seem to be independent of p44/42 MAPK and pFAK pathways.