Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB and both gene products undergo alternative splicing in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterised the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC variants differentially influenced clathrin knee conformation within assemblies, and clathrin lattices with neuronal CLC mixtures were more effective in membrane bending than those with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice deficient for nCLCa or nCLCb were defective in synaptic vesicle recycling. Mice with only nCLCb had a reduced synaptic vesicle pool compared to wild-type mice, while nCLCa-only mice had increased synaptic vesicle numbers. These findings highlight functional differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin function in neurons.