Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment under low rigidity is still unknown. Here we show that low rigidity of collagen gel down-regulates 1 integrin activation, clustering and FAKY397 phosphorylation, which is mediated by delayed raft formation. Moreover, overexpression of auto-clustered 1 integrin (V737N) but not constitutively active 1 integrin (G429N) rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer (FRET) to assess 1 integrin clustering, we have found that substratum rigidity between 58-386 pascal (Pa) triggers 1 integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of 1 integrin clustering enhances the interaction between 1 integrin, FAK and talin. Our results indicate that the contact with collagen fibrils is not sufficient for integrin activation. However, substratum rigidity is required for integrin clustering and activation. Taken together, our findings provide a new insight into the mechanosensing machinery and the mode of action for epithelial cells in response to their physical environment under low rigidity.