The route of pathogen contamination (from roots versus from leaves) of spinach leaves was investigated with a hydroponic cultivation system. Three major bacterial pathogens, Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes, were inoculated into the hydroponic solution, in which the spinach was grown to give concentrations of 10⁶ and 10³ CFU/ml. In parallel, the pathogens were inoculated onto the growing leaf surface by pipetting, to give concentrations of 10⁶ and 10³ CFU per leaf. Although contamination was observed at a high rate through the root system by the higher inoculum (10⁶ CFU) for all the pathogens tested, the contamination was rare when the lower inoculum (10³ CFU) was applied. In contrast, contamination through the leaf occurred at a very low rate, even when the inoculum level was high. For all the pathogens tested in the present study, the probability of contamination was promoted through the roots and with higher inoculum levels. The probability of contamination was analyzed with logistic regression. The logistic regression model showed that the odds ratio of contamination from the roots versus from the leaves was 6.93, which suggested that the risk of contamination from the roots was 6.93 times higher than the risk of contamination from the leaves. In addition, the risk of contamination by L. monocytogenes was about 0.3 times that of Salmonella enterica subsp. enterica serovars Typhimurium and Enteritidis and E. coli O157:H7. The results of the present study indicate that the principal route of pathogen contamination of growing spinach leaves in a hydroponic system is from the plant's roots, rather than from leaf contamination itself.