ABSTRACT: This work was performed to generate the data needed to set epidemiological cut-off values for minimal inhibitory concentrations (MICs) of ten antimicrobial agents against Vibrio parahaemolyticus determined using standardised broth microdilution protocols. Eight laboratories performed broth microdilution tests with incubation at 35°C for 16–20 h, and 7 also performed tests on the same isolates with incubation at 28°C for 24–28 h. Data were analysed by the ECOFFinder and normalised resistance interpretation algorithms. The cut-off values calculated for ceftazidime, florfenicol and trimethoprim/sulfamethoxazole, 1 µg ml–1, 1 µg ml–1 and 0.25/4.75 µg ml–1, respectively, were the same when calculated from data obtained at both temperatures. The cut-off values calculated from data obtained at 35°C and from data obtained at 28°C were 0.25 µg ml–1 and 0.5 µg ml–1 for enrofloxacin, 2 µg ml–1 and 4 µg ml–1 for gentamicin, 0.5 µg ml–1 and 1 µg ml–1 for oxolinic acid and 2 µg ml–1 and 1 µg ml–1 for oxytetracycline, respectively. The influence of the incubation temperature on MICs obtained was investigated by comparing MICs obtained at 35°C and 28°C for a specific antimicrobial agent with a particular isolate by an individual laboratory. Fifty-six percent of 1473 of these paired MIC values were identical while 38% differed from one another by not more than one dilution step. The data generated in this work will be submitted to the Clinical and Laboratory Standards Institute for consideration in their setting of internationally agreed-upon epidemiological cut-off values for V. parahaemolyticus that are essential for interpreting antimicrobial susceptibility testing data of this species.