The moisture aging effect and mechanism of asphalt binder during the in-service life of pavement were investigated by laboratory simulating tests. Pressure aging vessel (PAV) test simulating the long-term aging of binder during the in-service life of pavement was modified to capture the long-term moisture aging effect of binder. Penetration grade tests including penetration test, soften point test, and ductility test as well as Superpave^TM performance grade tests including viscosity test, dynamic shear rheometer test, and bending beam rheometer test were conducted to fully evaluate the moisture aging effect of binder. Fourier transform infrared spectroscopy test and Gel-permeation chromatography test were applied to provide a fundamental understanding of the moisture aging mechanism of binder. The results indicate that moisture condition can accelerate the aging of asphalt binder and shorten the service life of asphalt binder. The modified PAV test with moisture condition can well characterize the moisture aging properties of asphalt binder.