High temperature can induce the production of 2n gametes and aborted pollen during microsporogenesis in Populus canescens. However, the mechanism by which high temperature induces pollen abortion remains unknown. Here, pollen abortion was induced by exposing male flower buds of P. canescens to 38 and 41 °C; pollen morphology, meiotic abnormalities, defects of the meiotic microtubular cytoskeleton, and tapetum development were characterized, and expression analysis of the Actin gene was conducted. We found that the dominant meiotic stage, temperature, and duration of treatment significantly affected the percentage of high temperature-induced aborted pollen. Damaged spindle microtubules and depolymerized microtubular cytoskeletons were observed, which resulted in many lagging chromosomes at anaphase Ⅰ and Ⅱ, as well as aneuploid male gametes and micronuclei, generating aborted pollen grains. Tapetum disintegration was also delayed. However, the anther dehisced normally, and some viable pollen grains were released, suggesting that the delayed degradation of the tapetum was not responsible for pollen abortion. A significant reduction in PtActin gene expression was detected in treated cells, indicating that spindle actin was disrupted. The spindle actin appeared to protect cells against chromosome segregation errors during meiosis.