Heat shock protein family A (Hsp70) member 5 (HSPA5) is an endoplasmic reticulum chaperone, which regulates cell metabolism, particularly lipid metabolism. While HSPA5’s role in regulating cell function is well described, HSPA5 binding to RNA and its biological function in nonalcoholic fatty liver disease (NAFLD) is still lacking. In the present study, the ability of HSPA5 to modulate alternative splicing (AS) of cellular genes was assessed using Real-Time PCR on 89 NAFLD-associated genes. RNA immunoprecipitation coupled to RNA sequencing (RIP-Seq) assays were also performed to identify cellular mRNAs bound by HSPA5. We obtained the HSPA5-bound RNA profile in HeLa cells and peak calling analysis revealed that HSPA5 binds to coding genes and lncRNAs. Moreover, RIP-Seq assays demonstrated that HSPA5 immunoprecipitates specific cellular mRNAs such as EGFR, NEAT1, LRP1 and TGFß1, which are important in the pathology of NAFLD. Finally, HSPA5 binding sites may be associated with splicing sites. We used the HOMER algorithm to search for motifs enriched in coding sequence (CDs) peaks, which identified over-representation of the AGAG motif in both sets of immunoprecipitated peaks. HSPA5 regulated genes at the 5′UTR alternative splicing and introns and in an AG-rich sequence-dependent manner. We propose that the HSPA5-AGAG interaction might play an important role in regulating alternative splicing of NAFLD-related genes. This report is the first to demonstrate that HSPA5 regulated pre-RNA alternative splicing, stability, or translation and affected target protein(s) via binding to lncRNA and mRNA linked to NAFLD.