An imbalance in the synthesis of ribosomal proteins can lead to the disruption of various cellular processes. For mammalian cells, it has been shown that the level of the eukaryote-specific ribosomal protein eL29, also known as the one interacting with heparin/heparan sulfate, substantially affects their growth. Moreover, in animals lacking this protein, a number of anatomical abnormalities have been observed. Here, we applied next-generation RNA sequencing to HEK293 cells transfected with siRNAs specific for the mRNA of eL29 to determine what changes occur in the transcriptome profile with a decrease in the level of the target protein. We showed that an approximately 2.5-fold decrease in the content of eL29 leads to statistically significant changes in the expression of more than a thousand genes at the transcription level, without a noticeable effect on cell viability, rRNA level, and global translation. The set of eL29-dependent genes included both up-regulated and down-regulated ones, among which there are those previously identified as targets for proteins implicated in oncogenesis. Thus, our findings demonstrate that an insufficiency of eL29 in mammalian cells causes a significant reorganization of gene expression, thereby highlighting the relationship between the cellular balance of eL29 and the activities of certain genes.