The cytoskeleton is essential for mechanical signal transduction and autophagy. However, few studies have directly demonstrated the contribution of the cytoskeleton to mechanical stress-induced autophagy. We explored the role of the cytoskeleton in response to compressive force-induced autophagy in human cell lines. Inhibition and activation of cytoskeletal polymerization using small chemical molecules revealed that cytoskeletal microfilaments are required for changes in the number of autophagosomes, whereas microtubules play an auxiliary role in mechanical stress-induced autophagy. The intrinsic mechanical properties and special intracellular distribution of microfilaments may account for a large proportion of compression-induced autophagy. Our experimental data support that microfilaments are core components of mechanotransduction signals.