TNF is a major contributor on the pathogenesis of insulin resistance associated with obesity and inflammation by serine phosphorylating and degrading IRS1. Presently we further found that pretreatment with TNF inhibited insulin-induced phosphorylation of Akt2 greater than Akt1.Since lipid phosphatases SHIP2 and PTEN are negative regulators of insulin's metabolic signaling at the step downstream of PI3-kinase, we investigated the Akt isoform-specific properties of these phosphatases in the negative regulation following short- and long-term insulin treatment, and examined the influence of inhibition on the amelioration of insulin resistance caused by TNF in 3T3-L1 adipocytes. Adenovirus-mediated overexpression of WT-SHIP2 decreased the phosphorylation of Akt2 greater than Akt1 following insulin stimulation up to 15 min. Expression of a dominant-negative IP-SHIP2 enhanced the phosphorylation of Akt2 up to 120 min. On the other hand, overexpression of WT-PTEN inhibited the phosphorylation of both Akt1 and Akt2 following short-, but not long-term, insulin treatment. The expression of IP-PTEN enhanced the phosphorylation of Akt1 at 120 min and that of Akt2 at 2 min. Interestingly, the expression of IP-SHIP2, but not IP-PTEN, protected against the TNF inhibition of insulin-induced phosphorylation of Akt2, GSK3, and AS160, whereas both improved the TNF inhibition of insulin-induced 2-deoxyglucose uptake. The results indicate that these lipid phosphatases possess different characteristics according to the time and preference of Akt isoform-dependent signaling in the negative regulation of insulin's metabolic actions, whereas both inhibitions are effective in the amelioration of insulin resistance caused by TNF.