The spindle assembly checkpoint (SAC) has been established as an important mechanism of driving aneuploidy, which occurs at a high frequency in the colorectal tumorigenesis. Two important components of SAC are MAD1L1 and MAD2L1, which function together in an interactive manner to initiate the checkpoint signal. We hypothesize that genetic variants in the binding domains of MAD1L1 and MAD2L1 may modulate protein structures and eventually contribute to CRC susceptibility. A case-control study including 710 CRC cases and 735 controls was performed to examine MAD1L1 Arg558His and MAD2L1 Leu84Met's conferring susceptibility to CRC. Cytokinesis-block micronucleus cytome assays were applied to assess the effect of two functional variants on chromosomal instability (CIN). Significant associations with CRC risk were observed for MAD1L1 Arg558His (OR = 1.38,95% CI: 1.09-1.75) and MAD2L1 Leu84Met in a dominant model (OR = 1.48,95% CI: 1.09-2.01). Moreover, significant multiplicative gene-smoking interactions were found in MAD1L1 Arg558His (P = 0.019) and MAD2L184 Leu/Met (P = 0.016) to enhance CRC risk. Additionally, the frequencies of lymphocytic micro-nucleated binucleated cells for MAD1L1 Arg558His polymorphism were significantly different in the exposed group (P = 0.013), but not in the control group. The study emphasized that MAD1L1 Arg558His and MAD2L1 Leu84Met can significantly interact with smoking to enhance CRC risk, and the genetic effects of MAD1L1Arg558His on CIN need to be further clarified in follow-up studies.