Protein kinase D (PKD) isoforms are protein kinase C (PKC) effectors in diacylglycerol (DAG)-regulated signaling pathways. Key physiological processes are placed under DAG control by the distinctive substrate specificity and intracellular distribution of PKDs. Comprehension of PKDs’ roles in homeostasis and signal transduction requires further knowledge of regulatory interplay among PKD and PKC isoforms, analysis of PKC-independent PKD activation and characterization of functions controlled by PKDs in vivo. C. elegans and mammals share conserved signaling mechanisms, molecules and pathways Thus, characterization of C. elegans PKDs could yield insights into regulation and function that apply to all eukaryotic PKDs. C. elegans DKF-1 (D Kinase Family-1) contains tandem, DAG binding (C1) modules, a PH domain and S/T protein kinase segment, which are homologous with domains in classical PKDs. DKF-1 and PKDs have similar substrate specificities. PMA switches on DKF-1 catalytic activity in situ by promoting phosphorylation of a single amino acid T⁵⁸⁸, in the activation loop. DKF-1 phosphorylation and activation are unaffected when PKC activity is eliminated by inhibitors. Both phosphorylation and kinase activity of DKF-1 are extinguished by substituting A for T⁵⁸⁸, Q for K⁴⁵⁵ (“kinase dead”) or incubating with protein phosphatase 2C. Thus, DKF-1 is a PMA-activated, PKC-independent D kinase. In vivo, dkf-1 gene promoter activity is evident in neurons. Both dkf-1 gene disruption (null phenotype) and RNAi-mediated depletion of DKF-1 protein cause lower body paralysis. Targeted DKF-1 expression corrected this locomotory defect in dkf-1 null animals. Supra-physiological expression of DKF-1 limited C. elegans growth to ~60% of normal length.