Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in the recognition of microbial pathogens and host alarmins. Ligation to TLR4 initiates a signaling cascade that leads to inflammation. Polymorphisms in bovine TLR4 have been associated with Mycobacterium avium ssp. paratuberculosis (MAP) susceptibility and resistance, the cause of Johne\'s disease, and milk somatic cell score, a biomarker of mastitis. Although the contribution of TLR4 to recognition of bacterial lipopolysaccharide (LPS) has been well characterized, its role in MAP recognition is less certain. Clustered regularly interspaced short palindromic repeats-Cas9 mediated gene editing was performed to generate TLR4 knockout (KO) mammary epithelial cells to determine if TLR4 expression is involved in the initiation of the host inflammatory response to MAP cell lysate (5 and 10 µg/mL) and Escherichia coli LPS (5 µg/mL). The absence of TLR4 in KO cells resulted in enhanced expression of key inflammatory genes (TNFA and IL6), anti-inflammatory genes (IL10 and SOCS3), and supernatant cytokine and chemokine levels (TNF-α, IL-6, IL-10, CCL3) in response to the MAP cell lysate (10 µg/mL). However, in response to LPS, the KO cells showed reduced expression of key inflammatory genes (TNFA, IL1A, IL1B, and IL6) and supernatant cytokine levels (TNF-α, IL-6, CCL2, IL-8) as compared with unedited cells. Overall, these results confirm that TLR4 is essential for eliciting inflammation in response to LPS; however, exacerbated gene and protein expression in TLR4 KO cells in response to MAP cell lysate suggests a different mechanism of infection and host response for MAP, at least in terms of how it interacts with TLR4. These novel findings show potential divergent roles for TLR4 in mycobacterial infections, and this may have important consequences for the therapeutic control of inflammation in cattle.