Background: In recent years, titanium dioxide (TiO2) nanoparticles (NPs) have been widely used in various industries due to their favorable chemical properties, and their contamination of the environment has attracted much attention, especially to aquatic animals. Methods: Therefore, we assessed the impact of TiO2 NPs (5 mg/L) on the marine bivalve, pearl oyster (Pinctada fucata martensii), especially gill metabolism. Pearl oysters were exposed to seawater containing 5 mg/L TiO2 NPs for 14 days, followed by 7 days of recovery in untreated seawater. Gill tissues and hepatopancreatic tissues were sampled on days 0, 14, and 21 of the experiment named C0, E14, and R7, respectively. Results: Metabolomic analysis identified 102 significantly different metabolites (SDMs) on gills tissue in pearl oysters following exposure to TiO2 NPs (C0 vs. E14). Compared with group C0, group E14 had 76 SDMs (such as acetylcholine, itaconic acid, citric acid, and taurine) with higher concentrations and 26 (including L-arginine and isobutyryl-L-carnitine) with lower concentrations. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that these SDMs enriched 28 pathways, including glycine, serine, and threonine metabolism, neuroactive ligand–receptor interaction, and taurine and hypotaurine metabolism. In addition, 116 SDMs were identified in E14 and R7 pearl oysters. Compared with group E14, group R7 had 74 metabolites (such as acetylcholine, 6-phosphogluconic acid, isocitric acid, and itaconic acid) with higher concentrations and 42 (including uracil, glycerophosphocholine, N-Acetyl-D-glucosamine) with lower concentrations. The SDMs identified between E14 and R7 enriched 25 pathways, including the pentose phosphate pathway, glutathione metabolism, and citrate cycle (TCA cycle). In addition, analysis of the energy metabolism-associated enzymes revealed that exposure to TiO2 NPs reduced Ca2+/Mg2+-ATPase, Na+/K+-ATPase, and Total-ATPase activities. Conclusions: These findings suggested that TiO2 NPs may inhibit the energy metabolism function of gill and hepatopancreas of pearl oysters. Meanwhile, TiO2 NPs may affect the normal functioning of immune and osmoregulatory functions of pearl oysters gill and even may lead to oxidative stress and neurotoxicity. Therefore, this study may provide a reference for analyzing the bioadaptation of marine bivalves to TiO2 NPs and the potential negative effects of TiO2 NPs on bivalves.