Our previous study showed that persimmon tannin was the main active component being responsible for the anti-hyperlipidemic effect of persimmon extract, but the underlying molecular mechanisms were unclear. This study aimed to evaluate the effects of persimmon tannin on hepatic lipid accumulation in L02 cells and the underlying mechanisms, as well as the structure–activity relationships. Persimmon tannin significantly reduced L02 cellar lipid accumulation, similar effects were observed in the cells treated with its two characteristic structural subunits A-type ECG dimers and A-type EGCG dimer. Furthermore, the expression of miR-122 and miR-33b and their target genes were also suppressed by persimmon tannin, A-type ECG and A-type EGCG dimer. These results indicated that persimmon tannin improved hepatic steatosis through regulating miR-122 and miR-33b. The characteristic structural subunits A-type ECG and A-type EGCG dimer served as the basis for the inhibition of persimmon tannin on free fatty acid induced steatosis in L02 cells.