Helicobacter suis infection is an important cause of gastric disease in pigs and humans. In pigs, a H. suis infection has been shown to cause chronic gastritis and decreased daily weight gain. In addition, it has been associated with ulceration of the pars oesophagea of the stomach. Pigs and pork are considered to be the main source of infection for humans. H. suis is found in the majority of pigs worldwide, but little is known on the presence of other microorganisms in the stomach of these animals. In this study, we aimed at analyzing the porcine gastric microbiota and at investigating differences of the gastric microbiota between H. suis-positive and H. suis-negative pigs. Quantitative PCR was performed on pooled samples of biopsies taken from the 4 different regions of the stomach from slaughterhouse pigs to determine H. suis positivity. Subsequently, 6 H. suis-positive and 6 H. suis-negative animals were selected for further analysis. After amplification of 16S rRNA genes from the bacterial population, sequencing was performed using the Genome Sequencer Junior System (Roche 454 Life Sciences). Although the microbiota was diverse and differed between animals, the most frequently detected bacteria were Fusobacterium spp. (including a putative new species), Lactobacillus spp., Campylobacter spp. and Escherichia coli. The number of E. coli bacteria was higher in the stomach of H. suis-positive pigs compared to H. suis-negative pigs, whereas more Campylobacter jejuni bacteria were present in H. suis-negative animals. Further in vitro experiments showed that growth of both E. coli and C. jejuni is stimulated when co-incubated with H. suis bacteria. Interestingly, both E. coli and C. jejuni were able to suppress the growth of H. suis. Further research is needed to obtain a better insight into the interactions of H. suis with other bacteria and their effects on gastric health.