Background: Precursor B-cell acute lymphoblastic leukemia (B-ALL) is one of the most common types of leukemias in children. The majority of B-ALL patients are distinguished by chromosomal rearrangements; however, alternative splicing and epigenetic deregulations can also change the expression level of transcripts correlated with B-ALL. Therefore, the identification of prognostic and predictive biomarkers as well as the use of individualized treatments can help in B-ALL therapy. In this study, we performed an RNA-seq analysis to determine differentially expressed RNA transcripts in B-ALL. Methods: The RNA-seq data of 79 B-ALL and 14 non-malignant ITP (immune thrombocytopenic purpura) samples were obtained from the Gene Expression Omnibus (GEO) database. Moreover, RNA-seq was performed for Iranian patients with B-ALL to identify differentially expressed genes (DEGs). In order to experimentally validate the findings, the mRNA expression of SPRING1 (or C12orf49) was evaluated in bone marrow aspiration samples of B-ALL patients using quantitative reverse transcription-PCR. Results: Differential expression analysis revealed 920 downregulated and 1216 upregulated genes in B-ALL compared to ITP samples. Quantitative RT-PCR revealed the significant upregulation of SPRING1 (80%) in B-ALL patients. Functional enrichment analysis exhibited that SPRING1 was principally associated with lipopolysaccharide-mediated signaling pathways. Conclusion: Our results provided evidence for the involvement of SPRING1 in the B-ALL pathogenesis. However, further functional and clinical research is needed to understand its role in dysregulation of lipopolysaccharide-mediated signaling pathways in B-ALL.