We carried out shipboard observations in Funka Bay, Hokkaido, Japan, monthly or bimonthly from December 2015 to November 2016. We measured vertical profiles of isoprene, chlorophyll-a (chl-a), and other parameters from surface to bottom layer (about 95 m) near the center of the bay. We found substantial increases in isoprene concentration in the surface mixed layer from February to March during the peak of the spring diatom bloom, in the bottom layer from March to April after the peak of the bloom, and in the subsurface layer (below the surface mixed layer) in summer from July to August, where there were also substantial chl-a concentration maxima. We attribute the isoprene increases in the surface and subsurface layers to photosynthetic production of isoprene by the dominant phytoplankton in the spring bloom and in summer, and that in the bottom layer to dark production of isoprene by diatom aggregates that settled from the surface euphotic zone. We also measured isoprene production in laboratory incubation experiments. The in-situ production rates of isoprene per unit chl-a in the surface mixed layer in the spring bloom, in the dark bottom layer during the bloom, and in the subsurface layer in summer (0.82, 0.03-0.13, and 7.38 pmol (μg chl-a)-1 d-1 , respectively) were consistent with our incubation results. We believe that this is the first report focused on dark production of isoprene by diatoms; the production rate of isoprene under the dark condition ranged from 4% up to 16% of that by photosynthesis.