Cysteine string proteins (CSPs) are secretory vesicle chaperones that are important for neurotransmitter release. We have previously reported an interaction of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels that results in a tonic G protein inhibition of the channels. In this report we directly demonstrate that two separate regions of CSP associate with G proteins. The N-terminal binding site of CSP, which includes the J domain, binds Galpha subunits but not Galphabeta subunits whereas the C terminal binding site of CSP associates with either free Galphabeta subunits or Galphabeta in complex with Galpha. The interaction of either binding site of CSP (CSP1-82 or CSP83-198) with G proteins elicits robust tonic inhibition of N-type calcium channel activity. However, CSP1-82 inhibition and CSP83-198 inhibition of calcium channels occur through distinct mechanisms. Calcium channel inhibition by CSP83-198 (but not CSP1-82) is completely blocked by co-expression of the synaptic protein interaction site (synprint) of the N-type channel, indicating that CSP83-198 inhibition is dependent on a physical interaction with the calcium channel. These results suggest that distinct binding sites of CSP can play a role in modulating G protein function and G protein inhibition of calcium channels.