Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling, and PTP1B inhibitors have been seen as promising therapeutic agents against obesity and type 2 diabetes. Here we report that the marine natural product hyrtiosal, from the marine sponge Hyrtios erectus, has been discovered to act as a PTP1B inhibitor and to show extensive cellular effects on PI3K/AKT activation, glucose transport, and TGFbeta/Smad2 signaling. This inhibitor wad able to inhibit PTP1B activity in dose-dependent fashion, with an IC(50) value of 42 microM in a noncompetitive inhibition mode. Further study with an IN Cell Analyzer 1000 cellular fluorescence imaging instrument showed that hyrtiosal displayed potent activity in abolishing the retardation of AKT membrane translocation caused by PTP1B overexpression in CHO cells. Moreover, it was found that this newly identified PTP1B inhibitor could dramatically enhance the membrane translocation of the key glucose transporter Glut4 in PTP1B-overexpressed CHO cells. Additionally, in view of our recent finding that PTP1B was able to modulate insulin-mediated inhibition of Smad2 activation, hyrtiosal was also tested for its capabilities in the regulation of Smad2 activity through the PI3K/AKT pathway. The results showed that hyrtiosal could effectively facilitate insulin inhibition of Smad2 activation. Our current study is expected to supply new clues for the discovery of PTP1B inhibitors from marine natural products, while the newly identified PTP1B inhibitor hyrtiosal might serve as a potential lead compound for further research.