<p>Prostate cancer is one of the most common cancers in men, and 10–20% of cases progress to a hormone-resistant form, posing a major therapeutic challenge. The microsomal prostaglandin E2 synthase-1 (mPGES-1), a key enzyme in prostaglandin E2 biosynthesis from arachidonic acid, has been predicted as a therapeutic target in suppressing prostate cancer. However, the impact of mPGES-1 on the cellular lipidome has not been systematically investigated. Here, this study applied LC-MS/MS analysis and high-spatial single-cell mass spectrometry imaging (MSI) analysis using atmospheric pressure matrix-assisted laser desorption/ionization-MSI (AP-MALDI-MSI) to investigate the role of the mPGES-1 gene in altering the lipidome of hormone-resistant DU145 prostate cancer cells. LC-MS/MS results revealed significant downregulation of phosphatidylcholine (PC) and triglyceride (TG) species, and upregulation of ceramide species in KD (stable knockdown of the mPGES-1 gene) compared to controls. Upregulation of PCs is the signature of the tumor microenvironment of prostate cancer cells, and the upregulated ceramides are associated with programmed cell death. Consistent findings were observed in high-spatial single-cell MSI analysis for PC(16:0_16:0) and PC(18:0_18:1), which were significantly (<i>p</i> &lt; 0.001) downregulated in KD compared to controls, and also revealed a striking loss of metabolic heterogeneity of these PC molecules. The loss of metabolic heterogeneity due to mPGES-1 knockdown may reduce cancer cells’ adaptive capacity, suggesting mPGES-1 inhibition as a strategy to overcome metabolic plasticity in hormone-resistant prostate cancer. Thus, this study highlights that suppressing mPGES-1 can be a future therapeutic target in preventing the progression of hormone-resistant prostate cancer development.</p>