Position-specific (PS) carbon isotope compositions of light hydrocarbons such as propane and butane isomers (n-butane and i-butane) can provide a wealth of information on the history of natural gases in the subsurface reservoirs and other environments. For PS carbon isotope analysis of butane isomers, we have established a GC-pyrolysis-GC-isotope ratio mass spectrometry method with demonstrated accuracy. With this method, we analyzed PS δ¹³C values of butane isomers generated from the systematic laboratory pyrolysis experiments of three different kerogen types (I, II, and III) at temperatures of 310–430 °C with corresponding thermal maturity (Easy %R_o) ranging from 0.7 to 3.3. The observed evolution in the abundances of butane isomers can be interpreted and semi-quantitatively modeled based on the abundances of different CC bonds within the kerogens at low maturity and thermal degradation of butane isomers at high maturity. The δ¹³C values at the central sites of both nC₄ and iC₄ were heavier than those at the terminal positions, similar to our previous observations of propane. Their isotopic evolution with the maturity were controlled largely by kinetic isotope effects associated with breaking of different CC bonds during the generation and degradation of butane isomers. Kinetic Monte Carlo (kMC) simulations of n-butane generated from thermal cracking of model kerogens (I, II, and III) and an oil with a series of reactions (homolytic cleavage, β-scission, radical isomerization, H-abstraction, and termination by radical recombination) provided generally consistent results with the experimental observations, although the difference in PS δ¹³C values between the central and terminal positions are somewhat overestimated. On the other hand, the kMC simulation with homolytic cleavage and capping reactions alone produced significant deviations from the experimental results. Re-assessment of very limited data of PS δ¹³C values of natural butanes with our experimental and simulation results show that biodegradation significantly increased δ¹³C values at the central positions, not only of propane, but also of both butane isomers. This study lays a foundation and demonstrates the potential of PS isotope geochemistry of butane isomers to further improve our understanding of the sources, and geochemical and microbial processes of light hydrocarbons in the subsurface and other natural environments.