The dysregulation of exosomal microRNAs (miRNAs) plays a crucial role in the development and progression of cancer. This study investigated the role of a newly identified serum exosomal miRNA miR-4256 in gastric cancer (GC) and the underlying mechanisms. The differentially expressed miRNAs were firstly identified in serum exosomes of GC patients and healthy individuals using next-generation sequencing and bioinformatics. Next, the expression of serum exosomal miR-4256 was analyzed in GC cells and GC tissues, and the role of miR-4256 in GC was investigated byin vitroandin vivoexperiments. Then, the effect of miR-4256 on its downstream target genes HDAC5/p16INK4awas studied in GC cells, and the underlying mechanisms were evaluated using dual luciferase reporter assay and Chromatin Immunoprecipitation (ChIP). Additionally, the role of the miR-4256/HDAC5/p16INK4aaxis in GC was studied usingin vitroandin vivoexperiments. Finally, the upstream regulators SMAD2/p300 that regulate miR-4256 expression and their role in GC were explored usingin vitroexperiments. miR-4256 was the most significantly upregulated miRNA and was overexpressed in GC cell lines and GC tissues;in vitroandin vivoresults showed that miR-4256 promoted GC growth and progression. Mechanistically, miR-4256 enhanced HDAC5 expression by targeting the promoter of the HDAC5 gene in GC cells, and then restrained the expression of p16INK4athrough the epigenetic modulation of HDAC5 at the p16INK4apromoter. Furthermore, miR-4256 overexpression was positively regulated by the SMAD2/p300 complex in GC cells. Our data indicate that miR-4256 functions as an oncogene in GC via the SMAD2/miR-4256/HDAC5/p16INK4aaxis, which participates in GC progression and provides novel therapeutic and prognostic biomarkers for GC.