BsYetJ is a bacterial homolog of transmembrane BAX inhibitor-1 motif-containing (TMBIM) membrane protein that plays a key role in the control of calcium homeostasis. Being the only member in TMBIM protein family with characterized structures, BsYetJ has great potential to become a valuable model for TMBIM structure-function relation study. Previous results have revealed the coexistence of multiple conformational substates during calcium transportation, particularly in the pore-lining TM7 when embedded in lipid bilayer. This suggests a link between TM7 and calcium gating. Here, we analyze the local environment of BsYetJ TM7 in lipid nanodiscs, using site-directed spin labeling electron spin resonance. Our results uncover the water and lipid accessibility change of TM7 due to calcium binding and suggest a gate-forming region of TM7 toward the protein center. This results in the alignment of polar residues along channel pathway to facilitate ion transportation. Furthermore, we show that perturbations in TM7 conformational dynamics can enhance both channel conductance and open probability, as identified by double electron-electron resonance and electrophysiology. Together, these results provide insights into the role of TM7 in BsYetJ calcium gating and demonstrate the importance of conformational dynamics in protein function.