Ribonucleic acid interference (RNAi) has become an effective tool for studying gene function in a variety of cells. The objective of this study was to compare the efficiency of gene silencing when siRNA were introduced into bovine zygotes by microinjection (as done previously; Tesfaye D et al. 2007 Mol. Reprod. Dev. 74, 978-988) v. a novel method of transfection in terms of gene knockdown and embryo development. For microin-jection, in vitro-produced bovine zygotes (16 h post insemination) were randomly assigned to 1 of 3 groups over 2 experiments. In Experiment 1, E-cadherin siRNA was injected at 100 μM (n = 168) and compared with PBS-injected (n = 180) and noninjected controls (n = 152). In Experiment 2, E-cadherin siRNA was injected at 375 μM (n = 154) and compared with PBS-injected (n = 136) and noninjected controls (n = 151). Embryos were subsequently cultured in vitro until Day 7 (day of IVF = Day 0). For transfection, the zona pellucida was removed from in vitro-produced zygotes. Zona-free zygotes were randomly assigned to 1 of 4 groups (i) GAPDH (n = 67), (ii) scrambled (n = 66), (iii) E-cadherin (n = 69) siRNA treatments at 100 nM or (iv) nontransfected controls (n = 66). Zygotes were incubated in transfection medium with siRNA for 1 h at 39°C, cultured individually in the well-of-the-well system to Day 7. The proportion of zygotes undergoing cleavage and developing to the blastocyst stage was recorded, and Day 7 embryos were frozen individually for mRNA analysis. Data for mRNA expression were fitted to a general linear model, and developmental stages were tested using ANOVA. Microinjection of 100 μM E-cadherin siRNA had no effect on phenotype (P > 0.05). Injection of PBS or 375 μM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5%, 45.5%, and 62.9%, respectively) and blastocyst stage (39.0%, 32.5%, and 45%, respectively) compared with noninjected controls (P < 0.05). The mRNA abundance of the target gene was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 μM and 375 μM compared with control and PBS-injected groups (P < 0.05). Transfection with E-cadherin siRNA decreased development of 8-cell embryos (20.3 v. 53.0%, respectively) and blastocysts (7.2 v. 18.2%, respectively) compared with controls (P < 0.05). The mRNA relative abundance was not different between controls (nontransfected, or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100nM E-cadherin siRNA led to a 70% reduction in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P < 0.05). Zona removal and transfection resulted in decreased embryo development compared with microinjection (P < 0.05). However, transfection yielded more efficient gene silencing of E-cadherin mRNA with reduced embryo development compared with microinjection. This technique of gene silencing could improve the efficiency of gene function studies in early bovine embryogenesis.