The glass transition temperature T_g of biopreservative formulations is important for predicting the long-term storage of biological specimens. As a complementary tool to thermal analysis techniques, which are the mainstay for determining T_g, molecular dynamics simulations have been successfully applied to predict the T_g of several protectants and their mixtures with water. These molecular analyses, however, rarely focused on the glass transition behavior of aqueous trehalose solutions, a subject that has attracted wide scientific attention via experimental approaches. Important behavior, such as hydrogen-bonding dynamics and self-aggregation has yet to be explored in detail, particularly below, or in the vicinity of, T_g. Using molecular dynamics simulations of several dynamic and thermodynamic properties, this study reproduced the supplemented phase diagram of trehalose–water mixtures (i.e., T_g as a function of the solution composition) based on experimental data. The structure and dynamics of the hydrogen-bonding network in the trehalose–water systems were also analyzed. The hydrogen-bonding lifetime was determined to be an order of magnitude higher in the glassy state than in the liquid state, while the constitution of the hydrogen-bonding network exhibited no noticeable change through the glass transition. It was also found that trehalose molecules preferred to form small, scattered clusters above T_g, but self-aggregation was substantially increased below T_g. The average cluster size in the glassy state was observed to be dependent on the trehalose concentration. Our findings provided insights into the glass transition characteristics of aqueous trehalose solutions as they relate to biopreservation.