Phosphite (Phi)-based fungicides, such as the commercial product Phostrol™, are widely used in potato late blight control. However, the Phi translocation efficiency and the efficacy against pathogen are less discussed. In this study, the Phi concentration were quantified by high performance ion chromatography (HPIC) and the Phi translocation efficiency in potato tissues was evaluated using potato cultivar Russet Burbank with foliar application of the Phostrol solution both under greenhouse and field conditions. In the greenhouse trials, it was found that Phi was translocated from leaves to roots within 3 h and its concentration was significantly increased in the roots 24 h after the Phostrol application. In the field trials, the application rate of Phostrol affected the Phi translocation in potato tubers. To assess the efficacy of Phi against P. infestans, both the inhibition and infection tests were carried out. In the inhibition tests, three most common strains of P. infestans in Canada (US-8, US-23 and US-24) were inoculated on pea agar containing different levels of Phi. In the infection tests, both of detached leaves and whole tubers that received Phi were infected by the three strains of P. infestans. The in vitro tests indicated that the US-8 strain is the most tolerant whereas the US-23 strain is the most sensitive to Phi. Also, the in vivo tests demonstrated the dose-dependent translocation of Phi in potato leaves and tubers decreased the severity of infection by P. infestans. Moreover, potential defense mechanisms related to salicylic acid (SA) and jasmonic acid (JA) pathways that might be activated by Phi were also explored. Overall, the results of the study provided evidences that high Phi translocation efficiency encouraged late blight suppression in potato production.