Guava (Psidium guajava L.) is a tropical evergreen tree that tolerates a wide range of frost-free environments. In recent years, the American market demand for exotic and nutritious fruits, like guava, has been increasing, and, with a long harvest period, guava can be a potential alternative, high-value cash crop in the United States. However, the major limitation with commercializing guava cultivation in the United States is its low cold tolerance. In this article, we studied the physiology of freezing tolerance and cold acclimation in guava. Laboratory freeze–thaw tests (on leaves), shoot growth and leaf relative water content measurements, leaf anthocyanin content analyses, and leaf protein analyses were performed on nonacclimated and cold-acclimated guava cultivars Lucknow-49 and Ruby × Supreme. The leaf freezing tolerance (expressed as LT50 values) of nonacclimated tissues was ≈–2.5 °C and significantly enhanced to ≈–4.4 °C after an environmentally controlled cold acclimation regime for both cultivars. However, when compared based on actual injury sustained by leaves at various freezing temperatures in a freeze–thaw test, ‘Ruby × Supreme’ exhibited significantly less injury than ‘Lucknow-49’ at most temperatures. Growth and leaf relative water content reduced, whereas leaf anthocyanins accumulated during cold acclimation. Leaf protein analyses, which were performed after cold acclimation and drought stress, revealed that four proteins (69, 48, 23.5, and 17.4 kDa) accumulated in response to low temperatures, and two proteins (17.4 and 16 kDa) accumulated in response to drought stress. Antidehydrin immunoblots revealed that one common 17.4 kDa dehydrin accumulated in response to cold and drought stresses. Our data indicate that guava possesses leaf freezing tolerance, exhibits cold acclimation ability, and that ‘Ruby × Supreme’ leaves are relatively more freezing-tolerant than ‘Lucknow-49’ when compared up to –4 and –8 °C for nonacclimated and cold-acclimated tissues, respectively. Cold acclimation in guava appears to be a multifactorial process involving complex physiological and biochemical changes and also overlapping responses with drought stress.