In this study, a combination of RNAi and endoscopic transfer to the oviduct of synchronized heifers has been used to investigate the effect of suppression of Cx43 and E-cadherin on the development, mRNA and protein expression of bovine blastocysts cultured in vitro or in vivo. In vitro matured and fertilized bovine zygotes were randomly assigned to one of four groups namely: Connexin43 dsRNA-injected (n = 790), E-cadherin dsRNA-injected (n = 775), water-injected (n = 774), and noninjected controls (n = 652). Following 2 days in vitro culture, 4- and 8-cell stage embryos from each treatment group were used for culture in vitro or in vivo. About half of the 4-8-cell stage embryos from each treatment group were transferred to the oviduct of synchronized heifers, while the remainder were further cultured in vitro. Embryos from in vivo culture were flushed from recipients on the fourth day post transfer (= Day 7 post insemination). Blastocyst stage embryos from both culture systems were used for mRNA and protein expression analysis. Irrespective of treatment or culture conditions, microinjection resulted in a decline in the proportion of embryos reaching the blastocyst stage. Significantly, lower blastocyst development was observed in E-cadherin and water-injected embryos following in vivo culture compared to the noninjected controls, while intermediate results were obtained following injection with Cx43 dsRNA. Both mRNA and protein products of the target genes were suppressed but the efficiency of suppression of the target genes varied depending on the initial level of transcript abundance, which is known to be greatly affected by the culture environment.