The von Hippel-Lindau tumour suppressor protein (pVHL) controls distinct cellular responses ranging from targeting hypoxia inducible factor α (HIFα) subunits for degradation and promotion of chromosomal stability to the regulation of microtubule dynamics. pVHL is produced in mammalian cells as a long and a short isoform, derived from alternate translational initiation sites in a single Vhl mRNA. However, it is unclear whether these isoforms have different cell biological activities that may represent different tumour suppressor activities of pVHL. Through a knock-in strategy to mutate the first translational initiation site from methionine to leucine (M1L) we have genetically deleted the pVHL long protein isoform in mice, allowing dissection of isoform-specific functions of pVHL. Vhl(M1L/M1L) mice exhibit no obvious phenotypic abnormalities. While numerous pVHL-mediated activities, including degradation of HIFα transcription factors, are unaffected, microtubule dynamics are altered in primary cells derived from Vhl(M1L/M1L) mice to an extent similar to that seen following complete loss of pVHL function. We conclude that the microtubule-regulating function and the HIFα-regulating function of pVHL are separable activities mediated by different protein isoforms.