A novel single-reactor system having entrapped anaerobic microorganisms has been developed to co-produce H2 and CH4. pH is one of the key operating and environmental parameters affecting the performance of a bio-system. This work aimed to investigate the pH shock effects on the novel biohythane system. The experiments were suddenly changing the original cultivation pH value of 6 into 4, 5, 7 or 8 for 4 h. The results indicate that a short pH shock could be used to regulate H2/CH4 composition without notably affecting biogas yield and chemical oxygen demand (COD) removal. Peak biohythane production was obtained after the pH shock to 8, having H2/CH4 yields of 11.5 ± 1.6/44.8 ± 3.1 mL/g COD. During pseudo steady-state conditions of effective cultivation periods, the values of H2 content in biohythane and COD removal efficiency were in ranges of 20–39% and 71–79%, respectively. The significances and applications of the experimental results have been discussed. The novelty of this work is elucidating a less-discussed field-operation problem of pH perturbances for a newly-developed biohythane system.