Aim To evaluate potential acylation stimulating protein (ASP) resistance in both adipocytes and preadipocytes under the conditions which produce insulin resistance by 17β-estradiol on both receptor level and post-receptor level. Methods 3T3-L1 preadipocytes were induced differentiated and 0 mol/L (17β-estradiol-free DMEM/F12), 10-8 mol/L, 10-7 mol/L and 10-6 mol/L 17β-estradiol was added to cultured 3T3-L1 adipocytes and preadipocytes overnight. RT-PCR and flow cytometry were used to detect mRNA and cell surface expression of ASP receptor. Both non-17β-estradiol treated and 17β-estradiol treated 3T3-L1 cells were cultured with 5.0 μmol/L ASP for 4 hours. Then the cell proteins were extracted and the expressions of Gβ, Gαq/11, p-PKCα and p-PKC ζ were measured by Western Blot. Results High dose 17β-estradiol suppressed C5L2 mRNA and protein expression in 3T3-L1 adipocytes but not preadipocytes. At 10-8 mol/L, 17β-estradiol increased C5L2 mRNA and protein expression slightly in both adipocytes and preadipocytes (P>0.05, respectively). At 10-6 mol/L 17β-estradiol inhibited C5L2 mRNA and cell surface C5L2 expression by 40% (P<0.05) and 21% (P<0.05), respectively. After overnight incubation with 17β-estradiol (in adipocytes and preadipocytes), Gαq/11, Gβ, p-PKCα and p-PKCζ were downregulated in the presence of ASP treatment to a certain degree. In adipocytes, at 10-6 mol/L, 17β-estradiol inhibited the ASP-induced Gαq/11, Gβ, p-PKCα and p-PKCζ expression by 17%,23%,15% and 15% (P>0.05, respectively). Whereas high dose 17β-estradiol effectively blocked ASP-stimulated Gαq/11 significantly by 24% (P<0.05) in preadipocytes. However, 17β-estradiol counteracted ASP-stimulated Gαq/11, Gβ, p-PKCα and p-PKCζ to some extent. Conclusion 17β-estradiol induces ASP resistance in adipocytes and preadipocytes. ASP resistance may contribute to the physiological abnormalities associated with insulin resistance induced by 17β-estradiol.