tRNA fragments (tRFs) are small non-coding RNA molecules that are generated through the cleavage of tRNAs and have been implicated in various biological processes. Among the different types of tRFs, the 3′-tRFs have attracted considerable scientific interest due to their regulatory role in gene expression. In this study, we investigated the role of 3′-tRF-CysGCA, a tRF derived from cleavage in the T-loop of tRNACysGCA, in the regulation of gene expression in HEK-293 cells. Previous studies have shown that 3′-tRF-CysGCA is incorporated into the RISC complex and interacts with Argonaute proteins, suggesting its involvement in the regulation of gene expression. However, the general role and effect of the deregulation of 3′-tRF-CysGCA levels in human cells have not been investigated so far. To address this gap, we stably overexpressed 3′-tRF-CysGCA in HEK-293 cells and performed transcriptomics and proteomics experiments. Moreover, we validated the interaction of this tRF with putative targets whose levels were affected after 3′-tRF-CysGCA overexpression. Last, we investigated the implication of 3′-tRF-CysGCA in various pathways using extensive bioinformatics analysis. Our results indicate that 3′-tRF-CysGCA overexpression led to changes in the cell expression profile, and we identified multiple pathways that were affected by the deregulation of this tRF. Additionally, our reporter assays demonstrated that 3′-tRF-CysGCA directly interacted with TMPO and ERGIC1, leading to changes in their expression levels. Taken together, these findings suggest that 3′-tRF-CysGCA plays a significant role in the regulation of gene expression and highlight the potential importance of this tRF in cellular processes.