Numerous proteins display pleiotropic functions in different clinical contexts. However, the molecular mechanism underlying such effects is rarely understood. Speckle-type POZ protein (SPOP) is a typical example, exhibiting tumor-suppressing or tumor-promoting effects in different tumor types in accordance with different amino acid changes; specifically, two distinct sets of variants in SPOP are commonly found in subsets of prostate cancer and endometrial cancer patients. To comprehensively characterize the functional landscape of SPOP alteration, we performed a deep mutational screening (DMS), elucidating the functionality of 7,933 out of 8,228 possible single amino acid changes (96.4% coverage). Leveraging the observation that overexpression of human SPOP leads to yeast growth arrest, we assessed the functionality of each variant using a yeast proliferation assay. In addition, our approach combined long-read and short-read sequencing. Finally, our DMS model enables a clear distinction of likely-loss-of-function variants that are enriched in prostate cancers and reveals their differential characteristics in both protein structure and genetic assessments. These results demonstrate the utility of our approach in high-resolution mapping and amino acid-level interpretation of protein function.