Objective To explore the relationship between ERG4 gene overexpression and azole resistance in clinical Candida albicans strains. Methods The National Committee for Clinical Laboratory Standards (NCCLS)M27-A2 broth microdilution method was conducted to evaluate antifungal susceptibility of 34 clinical Candida albicans isolates in vitro. Total RNA was extracted from these Candida albicans strains and transcribed into cDNA. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of ERG4 gene. Statistical analysis was carried out by a two-sample t-test. Results The expression level of ERG4 mRNA was significantly higher in fluconazole-resistant than in -sensitive Candida albicans strains (4.20 ± 2.56 vs. 1.72 ± 1.33, t = 3.99, P < 0.05), higher in itraconazole-resistant than in -sensitive Candida albicans strains (3.60 ± 2.47 vs. 1.66 ± 1.61, t = 3.71, P < 0.05), and higher in voriconazole-resistant than in -sensitive Candida albicans strains (3.99 ± 2.72 vs. 2.07 ± 1.58, t = 2.91, P <0.05). Further more, increased ERG4 mRNA expression was also observed in isolates cross-resistant to all the three azole antifungal agents compared with those susceptible to all of them (4.49 ± 2.73 vs. 1.69 ± 1.82, t = 3.81, P < 0.05). Conclusions The overexpression of ERG4 gene may be associated with cross resistance to fluconazole, itraconazole and voriconazole in clinical Candida albicans strains, but its exact role is expected to be investigated through downregulation of the ERG4 gene.