The MADS-box transcription factors play essential roles in many physiological and biochemical processes of plants, especially in fruit ripening. Here, a tomato MADS-box gene, SlCMB1, was isolated. SlCMB1 expression declined with the fruit ripening from immature green to B + 7 (7 days after Breaker) fruits in the wild type (WT) and was lower in Nr and rin mutants fruits. Tomato plants with reduced SlCMB1 mRNA displayed delayed fruit ripening, reduced ethylene production and carotenoid accumulation. The ethylene production in SlCMB1-RNAi fruits decreased by approximately 50% as compared to WT. The transcripts of ethylene biosynthesis genes (ACS2, ACS4, ACO1 and ACO3), ethylene-responsive genes (E4, E8 and ERF1) and fruit ripening-related genes (RIN, TAGL1, FUL1, FUL2, LoxC and PE) were inhibited in SlCMB1-RNAi fruits. The carotenoid accumulation was decreased and two carotenoid synthesis-related genes (PSY1 and PDS) were down-regulated while three lycopene cyclase genes (CYCB, LCYB and LCYE) were up-regulated in transgenic fruits. Furthermore, yeast two-hybrid assay showed that SlCMB1 could interact with SlMADS-RIN, SlMADS1, SlAP2a and TAGL1, respectively. Collectively, these results indicate that SlCMB1 is a new component to the current model of regulatory network that regulates ethylene biosynthesis and carotenoid accumulation during fruit ripening.