Objectives: Emergent antimicrobial-resistant hypervirulent Klebsiella pneumoniae (hvKp) is an important public health issue. We aimed to investigate resistance mechanisms and hypervirulent traits among tigecycline-non-susceptible (TNS) K. pneumoniae clinical strains, focusing on one hvKp strain with in vivo evolution of tigecycline resistance. Methods: TNS K. pneumoniae strains causing invasive diseases in a medical centre in Taiwan between July 2015 and April 2018 were collected. Resistance mechanisms were determined and hvKp strains were defined as rmpA/rmpA2-carrying strains. Isogenic strains with and without tigecycline resistance were subjected to WGS and in vivo virulence testing. Further, site-directed mutagenesis was used to confirm the resistance mechanism. Results: In total, 31 TNS K. pneumoniae strains were isolated, including six hypervirulent strains. Tigecycline resistance mechanisms were mostly caused by overexpression of AcrAB and OqxAB together with up-regulation of RamA or RarA, respectively. One TNS hypervirulent strain (KP1692; MIC=6 mg/L) derived from its tigecycline-susceptible counterpart (KP1677; MIC=0.75 mg/L) showed acrAB overexpression. WGS revealed four genetic variations between KP1677 and KP1692. In addition, using site-directed mutagenesis, we confirmed that a 1 bp insertion in the ramA upstream region (RamR-binding site), leading to ramA and acrAB overexpression in KP1692, was responsible for tigecycline resistance. The in vivo virulence experiment showed that the TNS hvKp strain KP1692 still retained its high virulence compared with KP1677. Conclusions: hvKp strains accounted for 19.4% among TNS strains. We identified alterations in the ramA upstream region as a mechanism of in vivo tigecycline resistance development in an hvKp strain.