Histone deacetylase 6 (HDAC6) uniquely endows as tumor suppressor in hepatocellular carcinogenesis, but the underlying mechanisms leading to tumor suppression are not fully understood. To identify comprehensive microRNAs (miRNAs) regulated by HDAC6 in hepatocellular carcinogenesis, differential miRNA expression analysis of HDAC6-transfected Hep3B cells was performed. Using integrative analyses of publicly available transcriptome data and miRNA target prediction, we selected 5 candidate miRNAs and, through in vitro functional validation, showed that let-7i-5p specifically suppressed thrombospondin-1 (TSP1) in hepatocellular carcinoma (HCC). Ectopic expression of antisense let-7i-5p (AS-let-7i-5p) inhibited in vitro tumorigenesis of HCC cells. In addition, treatments of partially purified TSP1 from culture cell media (ppTSP1) and recombinant TSP1 (rTSP1) exhibited similar effects with AS-let-7i-5p treatment on the same HCC cells, whereas TSP1 neutralizing antibody treatment significantly attenuated these effects. Notably, treatments of HDAC6 plasmid, AS-let-7i-5p, ppTSP1 and rTSP1 significantly suppressed in vitro angiogenesis and metastatic potential of HCC cells, but the co-treatment of TSP1 antibody specific to CD47 binding domain successfully blocked these effects in the same cells. Furthermore, we demonstrated that recovery of HDAC6 elicited let-7i-5p suppression to de-repress TSP1 expression, and thereby it occupied CD47 receptor to block CD47-SIRPα mediated anti-phagocytosis of macrophage in HCC. We also observed that HCC-derived exosomal let-7i-5p suppressed TSP1 of recipient hepatocyte cells. Treatments of HDAC6 plasmid, AS let-7i-5p and rTSP1 suppressed tumor incidence as well as tumor growth rates in a spontaneous mouse HCC model. CONCLUSION: Our findings suggest that HDAC6-let-7i-5p-TSP1 regulatory pathway suppresses neoplastic and anti-phagocytic behaviors of HCC by interacting with cell surface receptor CD47 in HCC and neighboring cells of tumor microenvironment providing a novel therapeutic target for the treatment of liver malignancy and metastasis. This article is protected by copyright. All rights reserved.