Kinetic analysis was used to study the destabilization of mayonnaise by focusing on the fat crystals. Mayonnaise prepared from rapeseed oil and soybean oil was stored at temperatures ranging from −20 to −40 °C. The destabilization kinetic parameters were measured by observing oil separation over time. The destabilization rate constant, kd, increased with decreasing temperature. The highest value of kd was 1.28 × 10−3 min−1 at −40 °C for rapeseed oil mayonnaise (RoM) and the lowest was 1.95 × 10−6 min−1 at −20 °C for soybean oil mayonnaise (SoM). At each temperature, the kd value in RoM was higher than that in SoM. However, the order of destabilization, n, followed no specific pattern. The crystallization rate constant, Kc, and Avrami constant, n, were calculated using microscopic images of the fat crystals. The increase in crystallization kinetic parameters with decreasing temperature revealed changes in crystal behavior. Both the destabilization rate constant, kd, and the crystallization rate constant, Kc, depended on the temperature. This temperature dependency behavior showed a correlation between kd and Kc, suggesting that the destabilization rate depended on the rate of growth of fat crystals during the freeze-thawing of mayonnaise.