This study aimed to investigate the effect of ultrasound combined with calcium lactate (2%, w/v) treatment (U + Ca) on calcium permeation and firmness of cherry tomatoes. Calcium distribution and fruit pectin nanostructure were also analysed by transmission electron microscope (TEM) and atomic force microscopy (AFM), respectively. The firmness (31.45 N) was maintained when ultrasound energy density was 20 W/L for 15 min at 15 °C. The Ca content increased in U + Ca treated fruit. Meanwhile, the Peleg’s model could be used to express the change of solid gain in cherry tomatoes under ultrasound treatment at 15, 20, and 25 °C. According to the AFM results, the width (≥40 nm) and length (≥2 μm) of chelate-soluble pectin (CSP) and sodium carbonate-soluble pectin (SSP) chains with large frequency was observed in U + Ca treated fruit. Under desirable conditions (15 °C, 15 min, 20 W/L), ultrasound combined with calcium lactate could maintain the quality of cherry tomatoes.