The once vast longleaf pine (Pinus palustris Mill.) forest ecosystems in the southeastern U.S. have dwindled to small remnants, often lacking frequent prescribed fire required to realize maximum ecosystem services and benefits. Reintroducing fire to restore these ecosystems can be associated with increased risk of subcortical insect (Coleoptera: Curculionidae: Scolytinae) colonization and tree mortality, necessitating an understanding of how beetles and tree defenses respond to restoration and management with prescribed fire. We evaluated the impacts of various fire regimes and site conditions in longleaf pine forest stands on subcortical beetles and tree defenses using a long-term fire regime experiment in southwestern Georgia, U.S. By sampling trap catches of over 140,000 beetles across three fire regime treatments and two edaphic site types and measuring stand characteristics, we found that effects of fire exclusion and site type varied at the species level for bark beetle trap catches and altered stand conditions. We observed higher resin flow in longleaf pine trees in stands that had been fire-excluded for 17 years compared to frequently burned stands, but we did not observe differences in total monoterpene concentration among the fire regime treatments. However, we found higher total monoterpene concentrations in xeric than mesic sites, indicating site-level differences in resin properties. We additionally investigated short-term effects on resin quantity and quality following dormant and growing season prescribed burns in mature, dominant longleaf pine trees. There was a temporary increase in resin flow following the dormant season prescribed burn, no effect on resin flow following the growing season burn, and no differences in total monoterpene concentration following either season burn. These novel experiments and results detailed herein provides restoration guidance to managers and directions for future research on endangered longleaf pine ecosystems in the southeastern U.S.