Severe irinotecan‐induced toxicity is associated with UGT1A1 polymorphisms. However, some patients develop side‐effects despite harbouring a normal UGT1A1 genotype. As CYP3A4 is also an irinotecan‐metabolizing enzyme, our study aimed to elucidate the influence of the CYP3A4*20 loss‐of‐function allele in the toxicity profile of these patients. Three‐hundred and eight metastatic colorectal cancer patients treated with an irinotecan‐containing chemotherapy were studied. The presence of CYP3A4*20, UGT1A1*37 and UGT1A1*28 alleles was tested. Associations between these genetic variants and toxicity were evaluated. UGT1A1*28 was significantly associated with severe diarrhoea, neutropenia and asthenia (P = 0.002, P = 0.037 and P = 0.041, respectively). One patient with the UGT1A1*28/*37 genotype presented with grade IV neutropenia and lethal septic shock. One heterozygous UGT1A1 (*1/*28) patient also carried the CYP3A4*20 allele but did not develop toxicity. We confirm that UGT1A1*37 and UGT1A1*28 are associated with severe toxicity and suggest that the CYP3A4*20 allele does not play a role in irinotecan‐induced toxicity.