Neutral trehalase 1 (Nth1) from Saccharomyces cerevisiae catalyzes disaccharide trehalose hydrolysis and helps yeast to survive adverse conditions, such as heat shock, starvation or oxidative stress. 14-3-3 proteins, master regulators of hundreds of partner proteins, participate in many key cellular processes. Nth1 is activated by phosphorylation followed by 14-3-3 protein (Bmh) binding. The activation mechanism is also potentiated by Ca(2+) binding within the EF-hand-like motif. This review summarizes the current knowledge about trehalases and the molecular and structural basis of Nth1 activation. The crystal structure of fully active Nth1 bound to 14-3-3 protein provided the first high-resolution view of a trehalase from a eukaryotic organism and showed 14-3-3 proteins as structural modulators and allosteric effectors of multi-domain binding partners.