BACKGROUND: Candidemia caused by Candida glabrata is a serious fungal infection, and rising echinocandin resistance presents a significant clinical challenge. Understanding the drug susceptibility profiles, molecular epidemiology, and mechanisms underlying adaptive echinocandin resistance in C. glabrata is crucial. RESULTS: A total of 106 C. glabrata strains were isolated from blood cultures of 103 candidemia patients across three medical centers in eastern China. Transcriptome sequencing and whole-genome sequence analysis were used to explore the genomic characteristics of echinocandin-resistant strains. Multi-locus sequence typing (MLST) categorized the isolates into 11 sequence types (STs), with ST7 being the most prevalent (67.9%). Drug susceptibility testing revealed a fluconazole resistance rate of 21.7%, while non-wild-type rates for voriconazole, itraconazole, and posaconazole were 23.6%, 7.5%, and 6.6%, respectively. One isolate (Q2-2) was resistant to all three echinocandins. Two isolates were resistant to micafungin and anidulafungin, respectively. Compared to the echinocandin-sensitive strains, the expression of the Chitin synthetase 3 (CHS3) gene was significantly upregulated in echinocandin-resistant strains. Functional analysis of a CHS3-overexpressing strain (ATCC2001-CHS3-OE), generated through homologous recombination, confirmed echinocandin resistance. Conversely, a CHS3 knockout strain (Q2-2-CHS3Δ) exhibited susceptibility to echinocandins. CONCLUSIONS: Our findings suggest that CHS3 plays a critical compensatory role in echinocandin resistance in C. glabrata, offering a promising target for developing future antifungal strategies.