Abstract BACKGROUND Oxathiapiprolin, developed by DuPont, is the only commercial oxysterol‐binding protein inhibitor (OSBPI) of oomycete pathogens. Although the activity of oxathiapiprolin on some Pythium spp. and Phytopythium spp. has been reported, it has not been tested on many other species, and little is known about the mechanisms of Pythium spp. that are tolerant to it. RESULTS Oxathiapiprolin exhibited a strong inhibitory effect on mycelial growth of Phy . litorale , Phy . helicoides and Phy . chamaehyphon , with EC 50 values ranging from 0.002 to 0.013 μg mL −1 . It also showed good effectiveness against Py . splendens and two Py . ultimum isolates, with EC 50 values ranging from 0.167 to 0.706 μg mL −1 , but showed no activity against 14 other Pythium spp. Oxathiapiprolin provoked a slight upregulation of PuORP1 in Py . ultimum , but it did not lead to PaORP1‐1 or PaORP1‐2 overexpression in Py . aphanidermatum . Transformation and expression of PuORP1 , PaORP1‐1 or PaORP1‐2 in the sensitive wild‐type Phytophthora sojae isolate P6497 confirmed that either the PuORP1 , PaORP1‐1 or PaORP1‐2 was responsible for the observed oxathiapiprolin tolerance. CONCLUSION This study showed that oxathiapiprolin had excellent activity against Phytopythium spp. but displayed a differentiated activity against different Pythium spp. ORP1s in Pythium spp. are positively related to the tolerance of Pythium species to oxathiapiprolin. © 2020 Society of Chemical Industry