U1 snRNP (U1 small nuclear ribonucleoprotein) is a well-characterized splicing factor. Besides, when U1 snRNP binds close to a putative polyadenylation site, mostly located in introns, it prevents premature cleavage and polyadenylation and controls the length of most cellular mRNAs. On the other hand, U1 snRNP binding close to the 3´-end of some mRNAs, inhibits polyadenylation and, therefore, gene expression. The inhibition of polyadenylation by U1 snRNP is the basis of U1i (U1 snRNP-based inhibition), a technique used to inhibit gene expression. U1i consists of the expression of a U1 snRNP modified to interact with a target mRNA and inhibit target gene expression. U1i has been used to inhibit the expression of reporter or endogenous genes both in tissue culture and in animal models. Furthermore, combination of U1i and RNA interference (RNAi) results in synergistic increased inhibitions which allow the dose of inhibitors to be decreased whilst at the same time obtaining good inhibitions with fewer unwanted secondary effects. The combination of RNAi and U1i is of special interest for antiviral therapy, as a functional decrease of the expression of replicative viral RNAs may require high inhibition and the combination of two or more inhibitors should decrease the possibility of escape mutants resistant to treatment. In fact, a therapy with U1i combined with RNAi is currently being developed for the treatment of HBV infections. We believe that this review will clarify the hallmarks of U1i technology and will encourage many laboratories to use U1i for functional studies and therapeutic applications.