The tumor suppressor Bin1 was identified through its interaction with the N-terminal region of Myc which harbors its transcriptional activation domain. Here we show that Bin1 and Myc physically and functionally associate in cells and that Bin1 inhibits cell proliferation through both Myc-dependent and Myc-independent mechanisms. Bin1 specifically inhibited transactivation by Myc as assayed from artificial promoters or from the Myc target genes ornithine decarboxylase (ODC) and alpha prothymosin (pT). Inhibition of ODC but not pT required the presence of the Myc binding domain (MBD) of Bin1 suggesting two mechanisms of action. Consistent with this possibility, a non-MBD region of Bin1 was sufficient to recruit a repression function to DNA that was unrelated to histone deacetylase. Regions outside the MBD required for growth inhibition were mapped in Ras cotransformation or HepG2 hepatoma cell growth assays. Bin1 required the N-terminal BAR domain to suppress focus formation by Myc whereas the C-terminal U1 and SH3 domains were required to inhibit adenovirus E1A or mutant p53, respectively. All three domains contributed to Bin1 suppression of tumor cell growth but BAR-C was most crucial. These findings supported functional interaction between Myc and Bin1 in cells and indicated that Bin1 could inhibit malignant cell growth through multiple mechanisms.