This research investigated early hyphal development of cucurbit powdery mildew (Podosphaera xanthii) on leaves of cucumber (Cucumis sativus L.) acclimatized to different relative humidities to clarify the factors that limit it. Cucumber seedlings were acclimatized to a low (10%) or high (90%) relative humidity (RH) at 30 °C from germination. When the cotyledons had expanded and the first true leaf had begun to emerge, the plants were inoculated, and initial conidial invasion, hyphal development, and secondary haustorial formation were evaluated. The initial invasion of P. xanthii into the epidermis was slower on the low-RH leaves than on the high-RH leaves, suggesting that structural properties, such as greater leaf mass per area, physically limited the development of P. xanthii. The hyphal development and secondary haustoria of conidia that had successfully infected the epidermis were also slower on the low-RH leaves. These post-invasion behaviors indicate that conidial development was also suppressed by nonstructural properties of low-RH leaves such as nutrients, which correlate with nonstructural defense. The reduced leaf N content of the low-RH leaves may in part have inhibited the post-invasion behaviors of P. xanthii.