The biosynthetic pathway of colchicine, a modified isoquinoline alkaloid from Colchicum species, involves (1S)-autumnaline 1 as the precursor and O-methylandrocymbine 2 as a key intermediate that undergoes ring expansion to form the tropolone nucleus of colchicine 4. C-13 of 2 is preserved in the formyl group of the minor alkaloid N-formyldemecolcine 3. To elucidate the mechanistic details of the ring expansion step 2→3, this study aimed to determine the fate of the diastereotopic hydrogens at C-13 of 2 (corresponding to C-3 of autumnaline 1) by introducing tritium labels stereospecifically or randomly at C-3 of autumnaline 1, alongside 14C-labeled internal standards. We synthesized racemic (3RS)-[3-3H1]autumnaline 1a, stereospecific (3R)-[3-3H1]autumnaline 1c, and (3S)-[3-3H1]autumnaline 1d, administered them to Colchicum plants, and analyzed the 3H:14C ratios in N-formyldemecolcine 3 and its formyl group (as p-bromophenacyl formate). Results showed that the ring-expansion process generating the tropolone ring of N-formyldemecolcine 3 (and thus colchicine 4) involves the stereospecific removal of the HS hydrogen from C-13 of dienone 2, while the HR hydrogen is entirely retained.