Bacteria use the type VI secretion system (T6SS) to deliver toxic effectors into bacterial or eukaryotic cells during interbacterial competition, host colonization, or when resisting predation. Identifying effectors is a challenging task, as they lack canonical secretion signals or universally conserved domains. Here, we identify a protein domain, RIX, that defines a class of polymorphic T6SS cargo effectors. RIX is widespread in the Vibrionaceae family and is located at N-termini of proteins containing diverse antibacterial and anti-eukaryotic toxic domains. We demonstrate that RIX-containing proteins are delivered via T6SS into neighboring cells and that RIX is necessary and sufficient for T6SS-mediated secretion. In addition, RIX-containing proteins can enable the T6SS-mediated delivery of other cargo effectors by a previously undescribed mechanism. The identification of RIX-containing proteins significantly enlarges the repertoire of known T6SS effectors, especially those with anti-eukaryotic activities. Furthermore, our findings also suggest that T6SSs may play an underappreciated role in the interactions between vibrios and eukaryotes.