Si poisoning on Al-5Ti-B master alloys has been restraining the effectiveness of grain refinement of hypoeutectic Al-Si casting alloys for over 60 years, and yet the underlying mechanism of this phenomenon remains unclear. In this work, Si poisoning in Al-Si/Al-5Ti-B system was systematically investigated by combining state-of-the-art electron microscopy, first-principles calculations and thermodynamic calculations. Different from the common belief that silicides coat and therefore poison TiB2, this study demonstrates that the segregation of Si atoms at the TiB2/α-Al interface is likely the cause of Si poisoning. Silicide was found to be thermodynamically unfavorable to form even in an alloy with 10 wt.%Si. On the other hand, an appreciable amount of Si (5–20 at.%) was found to segregate in the TiAl3 two-dimensional compound (2DC) which is critical for triggering the nucleation of α-Al on TiB2. The formation of Ti-Si covalent bond within TiAl3 2DC disturbs its lattice and reduces its chemical interaction with α-Al, which both obstruct the epitaxial nucleation of α-Al and hence leads to Si poisoning. This study suggests that composition engineering of TiAl3 2DC and TiB2 with elements less attractive to Si could be a viable way to mitigate Si poisoning. Display omitted