MicroRNAs regulate gene expression primarily at the post-translational level and have been increasingly implicated in the pathogenesis of various neurological diseases, indicating an important role in the brain. However, their exact functional roles are often unclear, limiting their potential for clinical applications. MicroRNA-135a-5p has been linked to numerous neurological diseases, synaptic plasticity, and density of dendritic spines. Dendritic spines are small actin-rich protrusions along neuronal dendrites where excitatory synapses are located. Dendritic spine density is regulated by numerous factors, but spine initiation factors and the actin cytoskeleton are central for determining the density and morphology of spines. Here, we elucidated the effects of microRNA-135a-5p mimic or microRNA-135a-5p inhibitor on spine initiation factors expression and actin dynamics using B16F1 melanocytes. Our experiments confirmed the predicted binding of microRNA-135a-5p to spine initiation factor Gas7 but not to the other three tested spine initiation factors. Transfection of the microRNA-135a-5p mimic stabilized actin cytoskeleton observed by live cell imaging and fluorescence recovery of photobleaching approaches. Based on our results, changes are modest but probably suitable for finetuning cellular processes, such as dendritic spine formation or stabilization. The complexity of regulation makes it challenging to create a precise picture of the effects of individual microRNAs.