Uropathogenic Escherichia coli, the predominant causative agent of urinary tract infections, use type 1 pili to bind and invade bladder epithelial cells. Upon entry, the bacteria rapidly replicate and enter a complex developmental pathway ultimately forming intracellular bacterial communities (IBCs), a niche with biofilm‐like properties protected from innate defences and antibiotics. Paradoxically, bacteria within IBCs produce type 1 pili, an organelle thought only to be an extracellular colonization factor. Thus, we investigated the function of type 1 pili in IBC development. The cystitis isolate, UTI89, was genetically manipulated for conditional fim expression under control of the tet promoter. In this strain, UTI89‐tetR/P_tet fim, piliation is constitutively inhibited by the tetracycline repressor, TetR. Repression is relieved by anhydrotetracycline (AHT) treatment. UTI89‐tetR/P_tet fim and the isogenic control strain, UTI89‐tetR, grown in the presence of AHT, colonized the bladder and invaded the superficial umbrella cells at similar levels at early times in a murine model of infection. However, after invasion UTI89‐tetR/P_tet fim became non‐piliated and was unable to form typical IBCs comprised of tightly packed, coccoid‐shaped bacteria in contrast to the control strain, UTI89‐tetR. Thus, this work changes the extracellular colonization functional paradigm of pili by demonstrating their intracellular role in biofilm formation.