DIF-1 is a polyketide that induces Dictyoselium amoebae to differentiate as prestalk cells. We performed a global quantitative screen for phosphorylation changes that occur within the first minutes after addition of DIF-1 using a triple-label SILAC approach. This revealed a new world of DIF-1 controlled signaling: with changes in components of the MAPK and protein kinase-B signaling pathways, components of the actinomyosin cytoskeletal signaling networks and in a broad range of small GTPases and their regulators. The results also provide evidence that the Ca(2+)/calmodulin dependent phosphatase calcineurin plays a role in DIF-1 signaling to the DimB prestalk transcription factor. At the global level DIF-1 causes a major shift in the phosphorylation/dephosphorylation equilibrium toward net dephosphorylation. Interestingly, many of the sites that are dephosphorylated in response to DIF-1 are phosphorylated in response to extracellular cAMP signaling. This accords with studies that suggest an antagonism between the two inducers and also with the rapid dephosphorylation of the cAMP receptor that we observe in response to DIF-1 and with the known inhibitory effect of DIF-1 on chemotaxis to cAMP. All MS data are available via ProteomeXchange with identifier PXD001555.