Mechanical force is increasingly recognized as an important axis in regulation of T cell function. Recently, we showed that force elicits agonist-specific TCR-pMHC catch bond (where force counterintuitively prolongs bond lifetime), which provides a biophysical mechanism of force-triggered T cell Ca2+ flux (Liu et al., Cell, in press). Here, we performed steered molecular dynamics (SMD) simulation and single-bond lifetime experiments to investigate the molecular mechanism of TCR-pMHC catch bond. SMD simulation of 2C TCR-agonist dissociation showed that pulling TCR-pMHC bond leads to characteristic rotation of MHCI α1/α2 domain relative to TCR variable region, yielding multiple new atomic interactions (e.g., hydrogen bond) that are absent in crystal structure. Force-regulated bond lifetime measured on naïve 2C T cells validated two major predictions of such catch bond mechanism. (1) Pulling with a geometry that favors the relative domain rotation (by pulling pMHC β chain rather than c-terminal of heavy chain) produced a more pronounced catch bond; (2) a single point mutation on pMHC that abolishes new hydrogen bond formation eliminated catch bond and lowered EC50 (proliferation) by more than three orders of magnitude with only two fold decrease in affinity and a longer zero force lifetime. These data highlight a dynamic perspective of TCR-pMHC structure that reveals novel insights on TCR-pMHC interaction and T cell function.