Summary Candida glabrata emerged in the last decade as a common cause of mucosal and invasive fungal infection, in large part due to its intrinsic or acquired resistance to azole antifungals such as fluconazole. In C. glabrata clinical isolates, the predominant mechanism behind azole resistance is upregulated expression of multidrug transporter genes CDR1 and PDH1. We previously reported that azole‐resistant mutants (MIC ≥ 64 μg ml−1) of strain 66032 (MIC = 16 μg ml−1) similarly show coordinate CDR1‐PDH1 upregulation, and in one of these (F15) a putative gain‐of‐function mutation was identified in the single homologue of Saccharomyces cerevisiae transcription factors Pdr1–Pdr3. Here we show that disruption of C. glabrata PDR1 conferred equivalent fluconazole hypersensitivity (MIC = 2 μg ml−1) to both F15 and 66032 and eliminated both constitutive and fluconazole‐induced CDR1‐PDH1 expression. Reintroduction of wild‐type or F15 PDR1 fully reversed these effects; together these results demonstrate a role for this gene in both acquired and intrinsic azole resistance. CDR1 disruption had a partial effect, reducing fluconazole trailing in both strains while restoring wild‐type susceptibility (MIC = 16 μg ml−1) to F15. In an azole‐resistant clinical isolate, PDR1 disruption reduced azole MICs eight‐ to 64‐fold with no effect on sensitivity to other antifungals. To extend this analysis, C. glabrata microarrays were generated and used to analyse genome‐wide expression in F15 relative to its parent. Homologues of 10 S. cerevisiae genes previously shown to be Pdr1–Pdr3 targets were upregulated (YOR1, RTA1, RSB1, RPN4, YLR346c and YMR102c along with CDR1, PDH1 and PDR1 itself) or downregulated (PDR12); roles for these genes include small molecule transport and transcriptional regulation. However, expression of 99 additional genes was specifically altered in C. glabrata F15; their roles include transport (e.g. QDR2, YBT1), lipid metabolism (ATF2, ARE1), cell stress (HSP12, CTA1), DNA repair (YIM1, MEC3) and cell wall function (MKC7, MNT3). These azole resistance‐associated changes could affect C. glabrata tissue‐specific virulence; in support of this, we detected differences in F15 oxidant, alcohol and weak acid sensitivities. C. glabrata provides a promising model for studying the genetic basis of multidrug resistance and its impact on virulence.