The first record of tracheal mites, Acarapis woodi, in Japan was made in 2010. These mites have since caused serious damage to the colonies of Japanese honey bees, Apis cerana japonica. In the present study, to control the mites on Japanese honey bees with l-menthol, an agent used for European honey bees, Apis mellifera, we investigated (1) the seasonality of menthol efficacy, (2) the overwintering mortality of menthol-treated colonies, and (3) the menthol residue in honey under field conditions in cooperation with private beekeepers of Japanese honey bees. Seasonal menthol efficacy was tested by applying 30 g of l-menthol for 1 month in different seasons. Mite prevalence was measured by dissecting the honey bee thorax. Overwintering mortality was monitored during winter after checking the mite prevalence in autumn, and was compared with that of untreated colonies reported in our previous study. The residual level of menthol in honey was measured by GC–MS. The results showed that the menthol-treated colonies had a smaller rate of increase in mite prevalence than the untreated colonies. The effects of menthol were highest in March and April. The winter mortality was depressed by menthol treatment. Honey samples extracted from the menthol-treated colonies included 0.4 ppm of menthol residue on average. Our findings suggest that menthol treatment is effective for controlling the tracheal mites on Japanese honey bees.