Endoplasmic reticulum (ER) stress results from imbalances in unfolded/misfolded proteins, contributing to a wide variety of human diseases. To better understand the mechanisms involved in the cellular response to ER stress in cardiomyocytes, we previously conducted a genome-wide screening in an in vitro ER stress model of rat cardiomyocytes, which highlighted amino acid transporter heavy chain, member 2 (SLC3A2) as an important factor in ER stress. In the present study, we characterized the role of SLC3A2 during the unfolded protein response (UPR), as one of the primary pathways activated during ER stress. First, we confirmed the induction of Slc3a2 mRNA expression following treatment with various ER stress inducers in rat cardiomyocytes (H9C2) and neural cells (PC12). Knockdown of Slc3a2 expression with small interfering RNA (siRNA) revealed that the encoded protein functions upstream of three important UPR proteins: ATF4, ATF6, and XBP1. siRNA-mediated knockdown of both SLC3A2 and mammalian target of rapamycin 1 (mTOR1) revealed that mTOR1 acts as a mediator between SLC3A2 and the UPR. RNA sequencing was then performed to gain a more thorough understanding of the function of SLC3A2, which identified 23 highly differentially regulated genes between the control and knockdown cell lines, which were related to the UPR and amino acid transport. Notably, flow cytometry further showed that SLC3A2 inhibition also enhanced the apoptosis of rat cardiomyocytes. Taken together, these results highlight SLC3A2 as a complex, multifunctional signaling protein that acts upstream of well-known UPR proteins with anti-apoptotic properties, suggesting its potential as a therapeutic target for ER stress-related diseases.