Heroin dependence is a complex mental disorder resulting from interactions between genetic and environmental factors. Identifying the susceptibility genes of heroin dependence is the basis for understanding the pathogenesis of heroin dependence. Using a total gene expression microarray, we detected 924 differentially expressed gene transcripts in lymphoblastoid cell lines (LCLs) between 19 male heroin-dependent individuals and 20 male control subjects, including 279 upregulated and 645 downregulated gene transcripts in heroin-dependent individuals. We verified the reduced expression of the neuron-specific enolase gene (ENO2) in heroin-dependent individuals using real-time quantitative polymerase chain reaction and Western blot analysis. We further compared the allele and genotype frequencies of three single nucleotide polymorphisms (SNPs, rs11064464, rs3213433 and rs10849541) of the ENO2 gene between 532 male heroin-dependent individuals and 369 male controls. No significant differences in the allele or genotype frequencies of these three SNPs were detected between these two groups. Nevertheless, we identified a haplotype (T-C-G) derived from these three SNPs significantly underrepresented in heroin-dependent individuals compared with the control group (72.7% versus 75.9%, P < 0.032), while two other rare haplotypes (C-A-G and T-C-A) significantly overrepresented in heroin-dependent individuals compared with the control group (P < 0.001). Further study, however, did not detect significant differences of the plasma concentration of neuron-specific enolase between these two groups. Our data suggest that the ENO2 gene might be associated with heroin dependence, and reduced ENO2 gene expression may confer increased risk to heroin dependence.