Aim: The aim of this study was to investigate the effects of dietary supplementation of Rhodotorula mucilaginosa solid-state fermentation product (RSFP) on the laying performance, egg quality and intestinal microbial flora of hens. Methods and Results: In this study, 40-week-old Roman laying hens (n = 216) were randomly assigned to one of the four groups: the control (CON) group, fed 87Á5% basal diet +12Á5% fermentation substrate; the 0Á5% RSFP group, fed 87Á5% basal diet +12Á0% fermentation substrate +0Á5% RSFP; the 2Á5% RSFP group, fed 87Á5% basal diet +10Á0% fermentation substrate +2Á5% RSFP; and the 12Á5% RSFP group, fed 87Á5% basal diet +12Á5% RSFP. The effect of each treatment was analysed in six replicates of nine hens. The experimental period was 31 days, which included a 3-day adaptation period. After 31 days of feeding, one hen from each replicate was randomly selected and killed, and the jejunum and digesta in the cecum were collected for the determination of the intestinal morphology and microbial flora respectively. Daily egg mass in the 2Á5 and 12Á5% RSFP groups and egg production and feed conversion ratio in the 12Á5% RSFP group were higher than those in control group (P < 0Á05). The yolk colour was improved in hens fed RSFPsupplemented diets (P < 0Á05). Hens fed RSFP-supplemented diet showed a decrease in the relative abundances of Proteobacteria, Bacteroides, Helicobacteraceae, Helicobacter and Lachnospiraceae UCG-002, but an increase in the relative abundance of Prevotellaceae UCG-001 in the cecum (P < 0Á05). Conclusion: Dietary RSFP supplementation improved the laying performance, egg quality and intestinal microflora of hens. Significance and Impact of the Study: Dietary supplementing diet with Rhodotorula mucilaginosa solid-state fermentation product, which is rich in carotenoids, improved the yolk colour and increased the carotenoid content, thereby improving the intestinal health of hens. selenium and iodine (Nicolle et al. 2003;Kotrbacek et al. 2004;Svoboda et al. 2009). Furthermore, carotenoids can improve health by enhancing immunity (Sparks 2006). In the past, carotenoids used in the poultry industry have mostly been chemically synthesized. Recently, consumers concern about the safety of foods has increased; hence, interest in natural additives has grown (Karadas et al.