Wolbachia are common heritable endosymbionts that influence many aspects of ecology and evolution in various insects, yet Wolbachia-mediated intracellular metabolic responses to temperature stress have been largely overlooked. Here, we introduced the Wolbachia strain wLhui from the invasive Liriomyza huidobrensis (Blanchard) into a Drosophila Schneider 2 cell line (S2) and investigated the metabolite profile of wLhui-infected (S2_wLhui) and uninfected cell lines (S2_wu) under short-term exposure to either high (37°C), moderate (27°C), or low (7 and 17°C) temperatures. We find that Wolbachia infection, temperature stress, and their interactions significantly affect cellular metabolic profiles. Most significantly, when comparing the changes in metabolites between S2_wLhui and S2_wu, glycerophospholipids, amino acids, and fatty acids associated with metabolic pathways, microbial metabolism in diverse environments, and other pathways were significantly accumulated at either low or high temperatures. Our findings suggest Wolbachia-induced cellular physiological responses to short-term temperature stress, which may in turn affect the fitness and adaptive ability of its host as an invasive species.