HSP20 (HSPB6), one of small heat shock proteins (HSPs), is constitutively expressed in various tissues and has several functions. We previously reported that the expression levels of HSP20 in human hepatocellular carcinoma (HCC) cells inversely correlated with the progression of HCC, and that HSP20 suppresses the growth of HCC cells via the AKT and mitogen-activated protein kinase signaling pathways. However, the exact mechanism underlying the effect of HSP20 on the regulation of these signaling pathways remains to be elucidated. To clarify the details of this effect in HCC, we explored the direct targets of HSP20 in HCC using human HCC-derived HuH7 cells with HSP20 overexpression. HSP20 proteins in the HuH7 cells were coimmunoprecipitated with the p85 regulatory subunit and p110 catalytic subunit of phosphoinositide 3-kinase (PI3K), an upstream kinase of AKT. Although HSP20 overexpression in HCC cells failed to affect the expression levels of PI3K, the activity of PI3K in the unstimulated cells and even in the transforming growth factor-α stimulated cells were downregulated by HSP20 overexpression. The association of HSP20 with PI3K was also observed in human HCC tissues in vivo. These findings strongly suggest that HSP20 directly associates with PI3K and suppresses its activity in HCC, resulting in the inhibition of the AKT pathway, and subsequently decreasing the growth of HCC.