Restricted availability of retinoic acid (RA) in the testicular milieu regulates transcriptional activity of c-kit (KIT, CD117), which aids in the determination of spermatogonial stem-cell differentiation. The effect of RA on c-kit has been reported previously, but its mode of genomic action remains unresolved. We studied the molecular machinery guiding RA responsiveness to the c-kit gene using spermatogonial stem-cell line C18–4 and primary spermatogonial cells. A novel retinoic acid response element (RARE) positioned at –989 nucleotides upstream of the transcription start site (TSS) was identified, providing a binding site for a dimeric RA receptor (i.e. retinoic acid receptor gamma (RARγ) and retinoic X receptor). RA treatment influenced c-kit promoter activity, along with endogenous c-kit expression in C18–4 cells. A comprehensive promoter deletion assay using the pGL3B reporter system characterised the region spanning –271 bp and –1011 bp upstream of the TSS, which function as minimal promoter and maximal promoter, respectively. In silico analysis predicted that the region –1011 to +58 bp comprised the distal enhancer RARE and activators such as spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1), specificity protein 1 (SP1) and four E26 transformation-specific (ETS) tandem binding sites at the proximal region. Gel retardation and chromatin immunoprecipitation (ChIP) assays showed binding for RARγ, PU.1 and SP1 to the predicted consensus binding sequences, whereas GABPα occupied only two out of four ETS binding sites within the c-kit promoter region. We propose that for RA response, an enhanceosome is orchestrated through scaffolding of a CREB-binding protein (CBP)/p300 molecule between RARE and elements in the proximal promoter region, controlling germ-line expression of the c-kit gene. This study outlines the fundamental role played by RARγ, along with other non-RAR transcription factors (PU.1, SP1 and GABPα), in the regulation of c-kit expression in spermatogonial stem cells in response to RA.