Traditionally, hippocampal long-term potentiation (LTP) of synaptic strength requires Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and other kinases, whereas long-term depression (LTD) requires phosphatases. Here, we found that LTD also requires CaMKII and its phospho-T286-induced “autonomous” (Ca2+-independent) activity. However, whereas LTP is known to induce phosphorylation of the AMPA-type glutamate receptor (AMPAR) subunit GluA1 at S831, LTD instead induced CaMKII-mediated phosphorylation at S567, a site known to reduce synaptic GluA1 localization. GluA1 S831 phosphorylation by “autonomous” CaMKII was further stimulated by Ca2+/CaM, as expected for traditional substrates. By contrast, GluA1 S567 represents a distinct substrate class that is unaffected by such stimulation. This differential regulation caused GluA1 S831 to be favored by LTP-type stimuli (strong but brief), whereas GluA1 S567 was favored by LTD-type stimuli (weak but prolonged). Thus, requirement of autonomous CaMKII in opposing forms of plasticity involves distinct substrate classes that are differentially regulated to enable stimulus-dependent substrate-site preference. Display omitted •Autonomous CaMKII activity is required not only for LTP, but also for LTD•LTP stimuli (strong but brief) favor traditional substrate site phosphorylation•LTD stimuli (weak but prolonged) instead favor a distinct substrate class•Deciding factor in substrate choice is further Ca2+/CaM stimulation of CaMKII CaMKII and its “autonomous” activity, induced by T286-autophosphorylation, is a crucial mediator of long-term potentiation (LTP) of synaptic strength. In this study, Dell’Acqua, Bayer, and colleagues show that this CaMKII autonomy is also required for long-term depression (LTD), an opposing form of synaptic plasticity. These opposing functions involve stimulus-dependent differential substrate site selection on GluA1: S831 (a traditional substrate favored by LTP-type stimuli) versus S567 (a distinct substrate class instead favored by LTD-type stimuli).