Outbreaks of invasive infections, with high mortality rates, caused by multidrug-resistantCandida aurishave been reported worldwide. Although hotspot mutations inFKS1are an established cause of echinocandin resistance, the actual contribution of these mutations to echinocandin resistance remains unknown. Here, we sequenced theFKS1gene of a caspofungin-resistant clinical isolate (clade I) and identified a novel resistance mutation (G4061A inducing R1354H). We applied the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to generate a recovered strain (H1354R) in which only this single nucleotide mutation was reverted to its wild-type sequence. We also generated mutant strains with only the R1354H mutation introduced intoC. auriswild-type strains (clade I and II) and analyzed their antifungal susceptibility. Compared to their parental strains, the R1354H mutants exhibited a 4- to 16-fold increase in caspofungin minimum inhibitory concentration (MIC) while the H1354R reverted strain exhibited a 4-fold decrease in caspofungin MIC. In a mouse model of disseminated candidiasis, the in vivo therapeutic effect of caspofungin was more closely related to theFKS1R1354H mutation and the virulence of the strain than its in vitro MIC. The CRISPR-Cas9 system could thus aid in elucidating the mechanism underlying drug resistance inC. auris.