The objective of this study was to determine the roughage to concentrate (R:C) ratio with rain tree pod meal (RPM) supplementation on in vitro fermentation using gas production technique. The experiment design was a 6${ imes}$4 factorial arrangement in a CRD. Factor A was 6 levels of R:C ratio (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) and factor B was 4 levels of RPM (0, 4, 8 and 12 mg). It was found that gas kinetic, extent rate (c) was linearly increased (p<0.01) with an increasing level of concentrate while cumulative gas production (96 h) was higher in R:C of 40:60. In addition, interaction of R:C ratio and RPM level affected $NH_3-N$ and IVDMD and were highest in R:C of 0:100 with 0, 4 mg of RPM and 40:60 with 8 mg of RPM, respectively. Moreover, interaction of R:C ratio and RPM level significantly increased total volatile fatty acids and propionate concentration whereas lower acetate, acetate to propionate ratios and $CH_4$ production in R:C of 20:80 with 8 mg of RPM. Moreover, the two factors, R:C ratio and RPM level influenced the protozoal population and the percentage of methanogens in the total bacteria population. In addition, the use of real-time PCR found that a high level of concentrate in the diet remarkably decreased three cellulolytic bacteria numbers (F. succinogenes, R. flavefaciens and R. albus). Based on this study, it is suggested that the ratio of R:C at 40:60 and RPM level at 12 mg could improve ruminal fluid fermentation in terms of reducing fermentation losses, thus improving VFA profiles and ruminal ecology.