Mycobacterium tuberculosis ESAT‐6 (MtbESAT‐6) plays essential roles in pathogenesis. MtbESAT‐6 exhibits a unique pore‐forming activity (PFA) that is not found in its ortholog from non‐pathogenic Mycobacterium smegmatis (MsESAT‐6). Here, we characterized the differential PFAs and found that exchange of I25‐H26/T25‐A26 between two proteins reciprocally affected their PFAs. MtbESAT‐6(IH/TA) had ~ 40% reduction, while MsESAT‐6(TA/IH) fully acquired its activity similar to MtbESAT‐6. Mutations of A17E, K38T, N67L or R74Q on MtbESAT‐6(IH/TA) further reduced the activity, with MtbESAT‐6(IH/TA‐17) being the lowest. This study suggests I25‐H26 as the pH‐sensor essential for MsESAT‐6 to fully acquire the activity, while multiple residues contributed to MtbESAT‐6 PFA.