Kisspeptin (Kp) and its receptor (KISS1R) are essential for reproduction, with dysfunction in their activities leading to reproductive disorders, such as precocious puberty and hypogonadotrophic hypogonadism. Kp is synthesised and secreted within specialist nuclei of the hypothalamus, modulating the HPG axis. Kp is a 54 amino acid peptide, which can undergo proteolytic cleavage into smaller peptides, including Kp10, which retain biological activities. However, little is known about the functional and biological significance of these differential Kp peptide variants. Kp/KISS1R are expressed in a range of tissues, with tissue specific variation in the Kp peptide variant locally produced, suggesting that the Kp54 proteolytic cleavage may provide a means of modulating the tissue-specific functionalities of the Kp/KISS1R system. Our study therefore aimed to assess the effects of Kp10 and Kp54 on KISS1R-mediated downstream signalling in different cell types. We first utilised HEK293 cells transiently expressing the KISS1R. As an indicator of Gαq activation, calcium mobilisation via Fluo4-direct dye and confocal microscopy showed differences in maximum responses to Kp10 and Kp54, with Kp10 more potent than Kp54 (P<0.001, n=816). Time-dependent analysis of Kp10 and Kp54 showed a similar acute 5 min-activation of P-ERK, which was sustained for 60 min. Although not significant, a trend for enhanced Kp10-dependent ERK activation was observed at all time points. To explore tissue-dependent effects of Kp10 and Kp54, we switched to GnRH neuronal cell line (FCBN4 cells). A phosphokinase array revealed differential regulation of 10 phosphokinases by Kp10 and Kp54. Time-dependent analysis of two phosphokinase hits via Western blotting showed a trend for increased Kp54-dependent P-ERK at 10 min in comparison to Kp10. Analysis of Kp10 and Kp-54-dependent P-CREB also suggested differences in time course experiments. Our findings suggest differences in the biopotencies of Kp10 and Kp54, with biological significance remaining to be determined.