(1) Background: Age-related macular degeneration (AMD) is closely related with retinal pigment epithelial (RPE) cell dysfunction. Although the exact pathogenesis of AMD remains largely unknown, oxidative stress-induced RPE damage is believed to be one of the primary causes. We investigated the molecular mechanisms of pentraxin 3 (PTX3) expression and its biological functions during oxidative injury. (2) Methods: Using enzyme-linked immunosorbent assays and real-time reverse transcription-polymerase chain reaction, we analyzed mRNA and protein levels of PTX3 in the presence or absence of oxidative stress inducer, sodium iodate (NaIO 3), in primary human H-RPE and ARPE-19 cells. Furthermore, we assessed cell death, antioxidant enzyme expression, and AMD-associated gene expression to determine the biological functions of PTX3 under oxidative stress. (3) Results: NaIO 3 increased PTX3 expression, in a dose- and time-dependent manner, in H-RPE and ARPE-19 cells. We found phosphorylated Akt, a downstream target of the PI3 kinase pathway, phosphor- mitogen-activated protein kinase kinase 1/2 (ERK), and intracellular reactive oxygen species (ROS) were predominantly induced by NaIO 3. NaIO 3-induced PTX3 expression was decreased in the presence of phosphoinositide 3 (PI3) kinase inhibitors, ERK inhibitors, and ROS scavengers. Furthermore, NaIO 3 enhanced mRNA expression of antioxidant enzymes such as glucose-6-phosphate dehydrogenase ( G6PDH), catalase (CAT), and glutathione S-reductase ( GSR) in the control shRNA expressing RPE cells, but not in hPTX3 shRNA expressing RPE cells. Interestingly, NaIO 3 did not induce mRNA expression of AMD marker genes, such as complement factor I ( CFI), complement factor H ( CFH), apolipoprotein E ( APOE), and toll-like receptor 4 ( TLR4) in hPTX3 shRNA expressing RPE cells. 4) Conclusions: These results suggest that PTX3 accelerates RPE cell death and might be involved in AMD development in the presence of oxidative stress.