Updated project metadata. Translation initiation generally occurs at AUG codons in eukaryotes although it has been shown that non-AUG or non-canonical translation initiation can also occur. However, the evidence for non-canonical translation initiation sites (TISs) is largely indirect and based on ribosome profiling studies. Here, using a strategy specifically designed to enrich N-termini of proteins, we demonstrate that many human proteins are translated at non-canonical TISs. The large majority of TISs that mapped to 5’ untranslated regions were non-canonical and led to N-terminal extension of annotated proteins or translation of upstream small open reading frames (uORF). There has been little controversy on whether the corresponding amino acid to the start codon is incorporated at TIS or methionine is still incorporated. Notably, methionine was incorporated at almost all non-canonical TISs identified in this study. Comparison of the TISs determined through mass spectrometry with ribosome profiling data revealed that about two-thirds of the novel annotations were indeed supported by ribosome profiling data. The sequence orthology analysis and relative translation frequency analysis of non-canonical TISs over canonical ones suggests that those non-canonical TISs are not leaky but they can have certain biological functions. Overall, this study provides evidence of protein translation initiation at non-canonical TISs and indicates that further studies are required for elucidation of functional implications of such non-canonical translation initiation.