The gut microbiota has recently emerged as an important regulator of brain physiology and behaviourin animals, and ingestion of certain bacteria (probiotics) therefore appear to be a potential treatment formajor depressive disorder (MDD). However, some conceptual and mechanistical aspects need furtherelucidation.We therefore aimed at investigating whether the habitual diet may interact with the effect of pro-biotics on depression-related behaviour and further examined some potentially involved mechanismsunderlying the microbe-mediated behavioural effects.Forty male Sprague-Dawley rats were fed a control (CON) or high-fat diet (HFD) for ten weeks andtreated with either a multi-species probiotic formulation or vehicle for the last five weeks.Independently of diet, probiotic treatment markedly reduced depressive-like behaviour in the forcedswim test by 34% (95% CI: 22–44%). Furthermore, probiotic treatment skewed the cytokine production bystimulated blood mononuclear cells towards IFN, IL2 and IL4 at the expense of TNF and IL6. In addition,probiotics lowered hippocampal transcript levels of factors involved in HPA axis regulation (Crh-r1, Crh-r2 and Mr), whereas HFD increased these levels. A non-targeted plasma metabolomics analysis revealedthat probiotics raised the level of indole-3-propionic acid, a potential neuroprotective agent.Our findings clearly support probiotics as a potential treatment strategy in MDD. Importantly, theefficacy was not attenuated by intake of a “Western pattern” diet associated with MDD. Mechanistically,the HPA axis, immune system and microbial tryptophan metabolism could be important in this context.Importantly, our study lend inspiration to clinical trials on probiotics in depressed patients.