Objective To observe and compare the genomic and transcription level of protein phosphatase 1 regulatory subunit 16A (PPP1R16A) gene in human hepatocellular carcinoma (HCC) tissues and adjacent tissues, and to explore the effect of specific sliencing of PPP1R16A on the proliferation of HCC LM3 cells. Methods Quantitative reverse transcription PCR (qRT-PCR) was used to assess genomic and transcription level of PPP1R16A gene. The specific small interfering RNA (siRNA) of PPP1R16A was synthetized in vitro, and was transfected into HCC LM3 cells with liposome. The experiment was divided into the following three groups, namely, PPP1R16A-siRNA (si-16A) transfected group, non-specific siRNA (NC) transfected group and blank control group. The genomic and transcription level of PPP1R16A gene was detected by qRT-PCR. The expression of PPP1R16A protein was detected by Western blotting analysis. CCK-8 and clone formation assay were used to investigate the proliferation ability of transfected cells. Cell cycle was investigated by flow cytometry. Results The genomic level (P<0.001) and transcription level (P<0.001) of PPP1R16A gene in human HCC tissues were significantly increased compared with those in the adjacent liver tissues; and the genomic level was found significantly correlated with transcription level of PPP1R16A gene (P=0.015). The results of CCK-8 and clone formation experiment in vitro showed that the cell proliferation of si-16A group was significantly inhibited compared with NC group and blank control group (P<0.001). Flow cytometry showed that cell cycle was suppressed in si-16A group. Conclusion The genomic and transcription levels of PPP1R16A gene are increased in HCC tissues. The proliferation of HCC LM3 cells is suppressed by inhibiting the PPP1R16A gene transcription, which suggests that PPP1R16A gene functions as an oncogene in HCC.