Objective:To investigate inhibitory effect of lactate dehydrogenase-A (LDHA) short hairpin RNA (shRNA) on the expression of LDHA in human cholangiocarcinoma cell line Hucct-1 and observe the effect of LDHA down-expression on the proliferation of Hucct-1 cells. Methods: Three pairs of oligonucleotides targeting LDHA were cloned into linearized pGPU6/GFP/Neo eukaryotic expression vector after annealing. Hucct-1 cells were transfected with the recombinant plasmids that were identified by sequencing. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to evaluate the effect of RNA interference on the transcription of LDHA gene and the expression of LDHA protein respectively. Results: DNA sequencing confirmed that three recombinant plasmids were successfully constructed. The si-LDHA-1 and si-LDHA-3 showed higher interfering efficiency. Compared with that of negative control,the protein expression inhibition rates of si-LDHA-1 and si-LDHA-3 were (50 ± 3)% (P < 0.05) and(70 ± 2)%(P < 0.05). MTT assay showed that the proliferation of LDHA knocked-down Hucct1 cell (si-LDHA-3) was slower than negative control Hucct1 cell(si-NC),and there was significant difference in the absorbance value between transfected cells and negative control cells at 96 h after transfection(P < 0.05). Conclusion: The recombinant plasmid can effectively knockdown the expression of LDHA in Hucct1 cells,and two efficient interfering targets in LDHA gene are screened out. Inhibition of LDHA expression evidently results in inhibition of Hucct-1 proliferation.