Aggregation of insulin initiated from the monomeric form proceeds via the secondary pathway of fragmentation. It was interesting to find that glycerol had the potential to transform the secondary pathway of aggregation from fragmentation to heterogeneous nucleation in a concentration dependent manner. Such a change in the secondary pathway was manifested by a change in the fibrillar morphology, wherein, longer fibrils were formed in the presence of glycerol. Glycerol could inhibit all the major steps of insulin aggregation. The analysis of the kinetic traces suggested that the inhibitory effect was most significant on the primary pathways, although secondary nucleation and elongation were also inhibited. In fact, at higher glycerol concentrations, the primary pathways were inhibited to such an extent that the majority of the aggregation was now driven by the secondary pathways. Our data suggest that glycerol binds to the early intermediates in the insulin aggregation pathway, and inhibits them from forming the aggregation competent species capable of elongation. As higher order species are formed in the aggregation pathway, the relative stabilization rendered by glycerol diminishes due to the exclusion of glycerol from the interface.