An F-actin scaffold protein, afadin, comprises two splice variants called l-afadin (a long isoform) and s-afadin (a short isoform). It is known that in adult tissues, l-afadin is ubiquitously expressed while s-afadin is restrictedly expressed in brain. In cultured cortical neurons, l-afadin potentiates axonal branching whereas s-afadin blocks axonal branching by functioning as a naturally occurring dominant-negative isoform that forms a heterodimer with l-afadin. However, the temporal and spatial expression pattern of s-afadin during development or across multiple tissues and organs has not been fully understood. In this study, using Western blotting and RT-qPCR techniques and the fluorescent splicing reporters, we examined the detailed expression patterns of l- and s-afadin isoforms across various tissues and cell types, including rat organs at developmental stages, primary cultured neuronal and non-neuronal cells prepared from the developing rat brain, and in neurons in vitro generated from P19 embryonal carcinoma (EC) cells. Both mRNA and protein of s-afadin were abundantly expressed in various regions of rat neuronal tissues, and their expression dynamically changed during development in vivo. The expression of s-afadin was also detected in primary rat cortical neurons, but not in astrocytes or fibroblasts, and the neuronal expression increased during neuronal maturation in vitro. The dynamic alternative splicing event of afadin during development was successfully visualized with the newly developed fluorescent splicing reporter plasmids at a single cell level. Moreover, s-afadin was undetectable in undifferentiated EC cells, and the expression was induced upon differentiation of the cells into neurons. These data suggest that spatiotemporal and dynamic alternative splicing produces differential expression patterns of afadin isoforms and that alternative splicing of afadin is controlled by the neuron-specific regulator(s) whose activity is triggered and dynamically altered during neuronal differentiation and maturation.