The innate immune element, cathelicidin antimicrobial peptide (CAMP), is vital in the formation of the antimicrobial barrier in skin. CAMP production is increased during epidermal differentiation and enriched in the stratum corneum. We recently identified an endoplasmic reticulum (ER) stress-mediated sphingosine-1-phosphate (S1P)- dependent mechanism of CAMP synthesis. Interestingly, in this study, we found that S1P synthesized by an isoform of sphingosine kinase (SPHK), SPHK1, serves as a signal for CAMP synthesis; and conversely, another isoform SPHK2 likely has a suppressor role or no role in CAMP production. Pertinently, prior studies showed that physiological ER stress is essential for normal epidermal differentiation. We here demonstrate that: increased ER stress is evident in differentiated cultured keratinocytes (KC); 2) increases in both CAMP and S1P production depend upon differentiation level of KC (proliferated<early-<late-stage of differentiated KC); 3) expression of SPHK1 and SPHK2 is increased and decreased, respectively, during KC differentiation; and 4) dihydroS1P that is preferentially synthesized by SPHK2 does not increase CAMP production. Finally, overexpression of wild type, but not dominant negative SPHK2, suppresses CAMP production in both proliferated and differentiated KC. Our current study suggests that alterations of both SPHK1 and SPHK2 levels coordinately increase CAMP production during epidermal differentiation.