Microbial pathogens exploit the ubiquitin system to facilitate infection and manipulate the immune responses of the host. In this study, susceptibility to Yersinia enterocolitica and Yersinia pseudotuberculosis invasion was found to be increased upon overexpression of the deubiquitinating enzyme otubain 1 (OTUB1), a member of the ovarian tumour domain-containing protein family. Conversely, OTUB1 knockdown interfered with Yersinia invasion in HEK293T cells as well as in primary monocytes. This effect was attributed to a modulation of bacterial uptake. We demonstrate that the Yersinia-encoded virulence factor YpkA (YopO) kinase interacts with a post-translationally modified form of OTUB1 that contains multiple phosphorylation sites. OTUB1, YpkA and the small GTPase ras homologue gene family member A (RhoA) were found to be part of the same protein complex, suggesting that RhoA levels are modulated by OTUB1. Our results show that OTUB1 is able to stabilize active RhoA prior to invasion, which is concomitant with an increase in bacterial uptake. This effect is modulated by post-translational modifications of OTUB1, suggesting a new entry point for manipulating Yersinia interactions with the host.