Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear-enriched transcript 2 (NEAT2), is highly conserved among mammals and highly expressed in the nucleus. It was first identified in lung cancer as a prognostic marker for metastasis but is also associated with several other solid tumors. In hepatocellular carcinoma (HCC), MALAT1 is a novel biomarker for predicting tumor recurrence after liver transplantation. The mechanism of overexpression in tumor progression remains unclear. In the present study, we investigated the role of specificity protein 1/3 (Sp1/3) in regulation of MALAT1 transcription in HCC cells. The results showed a high expression of Sp1, Sp3 and MALAT1 in HCC vs. paired non-tumor liver tissues, which was associated with the AFP level (Sp1, r=7.44, P=0.0064; MALAT1, r=12.37, P=0.0004). Co-silencing of Sp1 and Sp3 synergistically repressed MALAT1 expression. Sp1 binding inhibitor, mithramycin A (MIT), also inhibited MALAT1 expression in HCC cells. In conclusion, the upstream of MALAT1 contains five Sp1/3 binding sites, which may be responsible for MALAT1 transcription. Inhibitors, such as MIT, provide a potential therapeutic strategy for HCC patients with MALAT1 overexpression.