ABSTRACT The second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) have been implicated in the regulation of flagellar motility in diverse eukaryotes, yet the specific contributions of PKA isoforms remain unclear. Moreover, the kinetoplastid PKA is insensitive to cAMP. Here, we investigated subcellular localisations of PKA subunits in Leishmania and study the flagellar waveform parameters in PKA deletion mutants. Leishmania mexicana has three PKA catalytic (LmxPKAC1, LmxPKAC2 and LmxPKAC3) and two PKA regulatory (LmxPKAR1 and LmxPKAR3) isoforms. Expansion microscopy revealed that LmxPKAR1 was localised between the paraflagellar rod and the axoneme, and LmxPKAC1, LmxPKAC2 depend on LmxPKAR1 for enrichment in the flagellum. LmxPKAC3 required LmxPKAR3 for colocalisation at the cell cortex. Deletion of LmxPKAR3 resulted in increased flagellar LmxPKAC3 signal and increased swimming speed. Conversely, deletion of LmxPKAC3 resulted in reduced flagellar beat frequencies and swimming speed. The absence of LmxPKAC1 resulted in the loss of high frequency tip-to-base waveforms, and consequently reduced swimming speed. LmxPKAC1 dissociated from cytoskeletons upon addition of guanosine, adenosine or inosine, consistent with a nucleoside-based PKA activation mechanism. These data suggest that this non-canonical PKA pathway regulates the motility of Leishmania through the modulation of flagellar waveforms.