Aflatoxin-B1 (AFB1), a hepatocarcinogenic mycotoxin, was demonstrated to induce the high rate of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) participate in the regulation of several biological processes in HCC. However, the function of miRNAs in AFB1-induced HCC has received a little attention. Here, we applied Illumina deep sequencing technology for high-throughout profiling of microRNAs in HepG2 cells lines after treatment with AFB1. Analysis of the differential expression profile of miRNAs in two libraries, we identified 9 known miRNAs and 1 novel miRNA which exhibited abnormal expression. KEGG analysis indicated that predicted target genes of differentially expressed miRNAs are involved in cancer-related pathways. Down-regulated of Drosha, DGCR8 and Dicer 1 indicated an impairment of miRNA biogenesis in response to AFB1. miR-34a was up-regulated significantly, down-regulating the expression of Wnt/β-catenin signaling pathway by target gene β-catenin. Anti-miR-34a can significantly relieved the down-regulated β-catenin and its downstream genes, c-myc and Cyclin D1, and the S-phase arrest in cell cycle induced by AFB1 can also be relieved. These results suggested that AFB1 might down-regulate Wnt/β-catenin signaling pathway in HepG2 cells by up-regulating miR-34a, which may involve in the mechanism of liver tumorigenesis.