Tigecycline is regarded as a last-resort treatment for carbapenem-resistant (CRKP) infections, but increasing numbers of tigecycline-resistant isolates have been reported. The tigecycline resistance mechanisms in CRKP are undetermined. This study aimed to elucidate the mechanisms underlying tigecycline resistance in 16 tigecycline- and carbapenem-resistant (TCRKP) isolates. Mutations in tigecycline resistance determinant genes , , , (A), (L), (X), (M), were assessed by PCR amplicon sequencing, and mutations in and (A) exhibited high prevalences individually (81%) and in combination (63%). Eight functional mutation profiles reducing tigecycline sensitivity were verified by plasmid complementation of wild-type and mutant Using a site-specific mutant, the most frequent RamR mutation, A19V (60%), had no significant effect on tigecycline susceptibility or the upregulation of and Two (A) variants with double frameshift mutations, type 1 and type 2, were identified; type 2 (A) is novel. A parent strain transformed with a plasmid carrying type 1 or type 2 (A) increased the tigecycline MIC by 8-fold or 4-fold, respectively. Synergistic effects were observed in strains harboring no gene and a mutated (A), with an 8-fold increase in the tigecycline MIC compared with that in strains harboring only mutated (A) being seen. Overall, mutations in the and (A) efflux genes constituted the major tigecycline resistance mechanisms among the studied TCRKP isolates. The identification of strains exhibiting the combination of a deficiency and widespread mutated (A) is concerning due to the possible dissemination of increased tigecycline resistance in .