A combined observational and numerical modeling study was conducted to clarify the effects of wind straining on estuarine stratification. Long-term mooring observations in the middle of Chesapeake Bay showed an asymmetric stratification response to along-channel winds. The stratification decreased under up-estuary winds. Under down-estuary winds, however, the stratification increased at moderate wind speeds but decreased at high wind speeds. In concert with numerical modeling, the mooring and ship-based survey data were analyzed to test the wind-straining mechanisms. In the middle and upper parts of the estuary, the straining of the density field by the vertical shear of the along-channel current dominates the stratification response, enhancing stratification under the down-estuary winds but reducing it under the up-estuary winds. A regime diagram was constructed to place the wind-induced stratification change in terms of the dimensionless Wedderburn number W: it is a linearly function of W for W > 0 (up-estuary winds) but a parabolic function of W for W < 0 (down-estuary winds). In the lower part of the estuary, however, the stratification response to the wind direction was opposite to that in the middle and upper Bay. The wind-driven lateral circulation was much stronger there, and the cross-channel straining of the density field by the vertical shear in the lateral currents drove the stratification response in the lower Bay. Therefore, both the along-channel and cross-channel straining regulate the estuarine stratification, and the net stratification change over a wind event is determined by the relative strength of along-channel and cross-channel straining.