The in vivo activities and conformational changes of ribosome recycling factor from Thermoanaerobacter tengcongensis (TteRRF) with 12 successive C-terminal deletions were compared. The results showed that TteRRF mutants lacking one to four amino acid residues are inactive, those lacking five to nine are reactivated to a similar or a little higher level than wild-type TteRRF, and those lacking ten to twelve are inactivated again gradually. Conformational studies indicated that only the ANS binding fluorescence change is correlated well with the RRF in vivo activity change, while the secondary structure and local structure at the aromatic residues are not changed significantly. Trypsin cleavage site identification and protein stability measurement suggested that mutation only induced subtle conformation change and increased flexibility of the protein. Our results indicated that the ANS-detected local conformation changes of TteRRF and mutants are one verified direct reason of the in vivo inactivation and reactivation in Escherichia coli.