This study aimed to evaluate whether preharvest or postharvest application of glycine betaine (GB) has the potential to improve fruit quality [fruit firmness (FF), size, skin color, soluble solids content (SSC), and titratable acidity (TA)] and susceptibility to storage disorders (peduncle browning, pitting, and decay) in ‘Lapins’ or ‘Regina’ sweet cherries, and to determine whether factors such as application frequency or timing impacted the efficacy of GB spraying. Adding 2 or 4 g·L−1 GB to hydro-cooling water (0 °C) as postharvest treatment did not affect fruit size, skin color, SSC, TA, peduncle browning, or pitting development; however, it did result in fruit softening and a low incidence of decay. GB applied preharvest at 2 or 4 g·L−1 once at 1 week before harvest (1WBH) was more effective for retaining FF and less peduncle browning and pitting compared with postharvest treatment. Increasing the preharvest GB application frequency from one time (1WBH or pit hardening) to three times (pit hardening, straw color, and 1WBH) enhanced FF and TA levels and resulted in lower pitting. The reduction in fruit size was observed for ‘Regina’, but not for ‘Lapins’. Changes in the contents of phosphorous (P), potassium (K), and magnesium (Mg) were unaffected by GB at harvest, whereas three GB sprays increased the total nitrogen (N) content. Compared with ‘Lapins’, ‘Regina’ allowed more calcium (Ca) uptake by GB and ultimately had firmer flesh. In conclusion, three preharvest applications of 4 g·L−1 GB showed great potential to improve quality attributes, to reduce the susceptibility to storage disorders, and to increase the Ca content of ‘Regina’ cherries.